Article

Neurogenic differentiation of murine and human adipose-derived stromal cells

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  • K-Cell Consulting, San Diego, CA, United States
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Abstract

The identification of cells capable of neuronal differentiation has great potential for cellular therapies. We examined whether murine and human adipose-derived adult stem (ADAS) cells can be induced to undergo neuronal differentiation. We isolated ADAS cells from the adipose tissue of adult BalbC mice or from human liposuction tissue and induced neuronal differentiation with valproic acid, butylated hydroxyanisole, insulin, and hydrocortisone. As early as 1-3 h after neuronal induction, the phenotype of ADAS cells changed towards neuronal morphology. Following neuronal induction, muADAS cells displayed immunocytochemical staining for GFAP, nestin and NeuN and huADAS cells displayed staining for intermediate filament M, nestin, and NeuN. Following neuronal induction of murine and human ADAS cells, Western blot analysis confirmed GFAP, nestin, and NeuN protein expression. Pretreatment with EGF and basic FGF augmented the neuronal differentiation of huADAS cells. The neuronal differentiation of stromal cells from adipose tissue has broad biological and clinical implications.

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... Most studies on neural differentiation of MSCs were carried out with human and rodent cells [2,[5][6][7][8][9][10][11][12][13]. In veterinary medicine, dogs are interesting for the development of novel regenerative treatments, and in addition to benefiting canine patients, these therapies might show translational potential as dogs could be a highly interesting model of human neurological disorders. ...
... In the present study, we confirmed that canine adipose tissue-derived MSCs are capable of neural differentiation in vitro and, furthermore, explored which neural induction medium is the most suitable for the neural differentiation of canine ASCs. In previous studies, rat and human multipotent mesenchymal stromal cells were shown to transdifferentiate into neural phenotypes by exposing these cells to a variety of neurogenic inductors, such as β-mercaptoethanol, butylated hydroxyanisole, potassium chloride (KCl), valproic acid, and forskolin [2,5,8,11,12]. Alternative methods to the chemical differentiation of human and canine MSCs into a neural lineage involves the addition of growth factors such as bFGF, EGF, neuroblast factor (N2), B27 supplement, and retinoic acid [7,13,15,22]. ...
... Differentiated rat and human multipotent mesenchymal stromal cells were shown to express mature neural markers, such as GFAP, MAP2, TUBB3, and neuron-specific enolase (NSE). Such cells also possess voltage-gated calcium channels and the ability to upregulate the glutamate receptor [2,5,8,11,12]. In the present study, after 24-hour preconditioning in predifferentiation medium STIM1 and subsequent incubation in NIMa differentiation medium for 3 or 9 days, cells expressed proteins characteristic for mature neurons and astrocytes. ...
Article
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Background: The ability of adipose tissue-derived multipotent mesenchymal stromal cells/mesenchymal stem cells (ASCs) to differentiate in neural lineages promises progress in the field of regenerative medicine, especially for replacing neuronal tissue damaged by different neurological disorders. Reprogramming of ASCs can be induced by the growth medium with neurogenic inductors and specific growth factors. We investigated the neural differentiation potential of canine ASCs using several growth media (KEM, NIMa, NIMb, NIMc) containing various combinations of neurogenic inductors: B27 supplement, valproic acid, forskolin, N2-supplement, and retinoic acid. Cells were first preconditioned in the pre-differentiation neural induction medium (mitogenically stimulated; STIM1), followed by the induction of neuronal differentiation. Results: After 3, 6, and 9 days of neural induction, elongated neural-like cells with bipolar elongations were observed, and some oval cells with light nuclei appeared. The expression of neuronal markers tubulin beta III (TUBB3), neurofilament H (NF-H), microtubule-associated protein-2 (MAP2), and glial fibrillary acidic protein (GFAP) was observed using immunocytochemistry, which confirmed the differentiation into neurons and glial cells. Flow cytometry analysis showed high GFAP expression (between 70 and 90% of all cells) after cells had been growing three days in the neural induction medium a (NIMa). Around 25% of all cells also expressed adult neuronal markers NF-H and MAP2. After nine days of ASCs differentiation, the expression of all neural markers was reduced. There were no differences between the neural differentiation of ASCs isolated from female or male dogs. Conclusions: The differentiation repertoire of canine ASCs extends beyond mesodermal lineages. Using a defined neural induction medium, the canine ASCs differentiated into neural lineages and expressed markers of neuronal and glial cells, and also displayed the typical neuronal morphology. Differentiated ASCs can thus be a source of neural cellular lineages for the regenerative therapy of nerve damage and could be useful in the future for therapy or the modelling of neurodegenerative diseases.
... Stem cells have great promise for tissue repair Regenerative medicine, and endothelial progenitor cells (EPCs) play an essential role in ischemic neovascularization. Adiposederived mesenchymal stem cells (AD-MSCs) could be one of the cellular sources for medical applications (2,3). Several studies, considered AD-MSCs in vitro because of their potential for direct differentiation into human heart cells and applied to the ischemic heart. ...
... To overcome the problem of ROS instability in measurement, malonaldehyde (MDA), a stable end product of lipid peroxidation, is often used as a marker of ROS production. Anti-oxidant scavenging systems defend organisms against ROS and inhibit oxidative damage [3]. The catalase (CAT), glutathione peroxidase (GSH), and total anti-oxidant capacity (TAC) of plasma are used as a scale of an organism's ability to defend against ROS. ...
Article
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Objectives: Background: Impaired coronary blood flow causes cardiac ischemia. Cellular therapy is a new approach to the treatment of myocardial ischemia. This study aimed to investigate the effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) conditioned with vasopressin on oxidative stress, perivascular collagen, and angiogenesis caused by myocardial infarction (MI) in rats. Materials and methods: We divided 40 male albino Wistar rats into 4 groups; Control group; No intervention; in experimental groups, after it generated induced MI on models, it divided into three groups: Vehicle group (150 μl of cell-free culture medium received); ASC-MI group (6× 106 AD-MSC received) and AVP-ASC-MI group (received 6 × 106 AD-MSC conditioned with 10 nM vasopressin). Then, histologic parameters and anti-oxidant enzymes were evaluated 7 days post-MI cell injection. Results: Arterial muscle diameter improved and collagen deposition around the coronary arteries decreased in cell-received groups compared with the vehicle group. Malondialdehyde (MDA), catalase (CAT), (GSH) Glutathione, and Total Anti-oxidant Capacity (TAC) parameters were not significantly different between the cells received groups compared with the vehicle group. But the Catalase (CAT) parameter in the ASC-MI group had a significant increase from the control group. Conclusion: We prepared direct evidence that intramyocardial injection of AD-MSCs reveals the positive cardiac remodeling post-MI in rats, and these useful effects can be more enhanced by administrating injection of conditioned ADSCs with vasopressin.
... Furthermore, they also hold mesodermal and ectodermal differentiation features. (Gronthos et al. 2001;Halvorsen et al. 2001;Sen et al. 2001;Erickson et al. 2002;Safford et al. 2002;Zuk et al. 2002;Ashjian et al. 2003). In the present study, isolated hASCs displayed distinctive mesenchymal stem cell phenotypes and expressed stem cell markers reported in other human MSC sources previously (Pittenger et al., 1999;Zuk et al., 2002;Gimble and Guilak, 2003;Katz et al., 2005;Strem et al., 2005;Kocaoemer et al., 2007;Bieback et al., 2009). ...
... Comparative studies of ASCs and MSCs from other sources recommended that ASCs held a better potential for neural differentiation Mostafavi et al., 2014). Various protocols used to convert hASCs into neural cells include exposure of butylated hydroxyanisole and dimethyl sulfoxide (Zuk et al. 2002;Safford et al. 2002), a cocktail of butylated hydroxyanisole, insulin, hydrocortisone and valproic acid (Saffford et al., 2002;Guilak et al. 2006) or a cocktail of indomethacin, insulin and isobutylmethylxanthine (Ashijan et al., 2003;Fujimura et al., 2005;Ning et al., 2006). Agents that increase intracellular cAMP levels De Ugarte et al. 2003), 5-azacytidine (Kang et al., 2003;Lee et al., 2008), mitogen bFGF and EGF together with forskolin (Nagase et al., 2007;Jang et al., 2010, Zavan et al., 2010Razavi et al., 2012), various glial growth factors (Kingham et al., 2007), and brain-derived neurotrophic factor (BDNF) with retinoic acid (Anghileri et al., 2008) could also induce neural differentiation of hASCs in vitro. ...
Conference Paper
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Human adipose stem cells (hASCs) among other stem cells offer a glimpse of possibilities in neurodegenerative disease treatment. By that fact, researchers have been developing neural transdifferentiation methods of hASCs over the last decades. Various types of inducing agents were applied to improve the protocol. NO-cGMP signaling pathway is known to play a vital role in regulation of cell growth, survival, differentiation, proliferation, migration, axon guidance and many other processes. cGMP, as the mediator, helps support NO regulation of cell survival, differentiation and neuroprotective regulation. Previous research demonstrated that applying NO donor agents in neural differentiation medium could convert hESCs into neural-like cells. However, neural differentiation through this specific signaling pathway on hASCs has not yet been investigated. In this study, 8 Bromo-cyclic GMP (8Br-cGMP) was added to neural induction cocktails to improve the neural transdifferentiation efficiency of hASCs. Results demonstrated that hASCs under this condition exhibited higher expression of neural genes and could further differentiate into mature neuronal and glial cells. Our findings highlighted that differentiated hASCs displayed neuronal progenitor profile, yet remained their plasticity as they can differentiate toward glial cells. Future studies employing in vivo transplantation models and their underlying mechanisms are warranted.
... AdSCs have been reported to be able to differentiate only into cells belonging to the mesoderm, such as osteoblasts, adipocytes, and myoblasts, and their differentiation direction is generally considered to be limited. However, in recent years, differentiation into endoderm-and ectoderm-derived cells (e.g., nerve cells, etc.) has been confirmed [4][5][6][7]. In addition, AdSCs promote cell proliferation, anti-inflammation, and angiogenesis by secreting cytokines [8][9][10][11]; thus, these cells show promising applications in cellular therapy. ...
Article
Full-text available
Adipose-derived stem cells (AdSCs), a type of mesenchymal stem cell, are expected to be applicable to regenerative medicine and cellular delivery systems. The maintenance of cell multipotency and control of the differentiation direction are important for these applications. However, the differentiation direction of these cells is widely believed to depend on the physical properties of their scaffold. In this study, we explored whether the multipotency of AdSCs, that is, their ability to differentiate into multiple cells, is maintained when they are removed from injectable polymer (IP) hydrogels with various degrees of cross-linking and induced to differentiate into osteoblasts and adipocytes. We confirmed that AdSCs cultured in IP hydrogels maintained an undifferentiated state. However, their differentiation into osteoblasts and adipocytes cannot be ensured; specifically, the multipotency of AdSCs may decrease when they are cultured in IP hydrogels. When cultured in an IP hydrogel with extreme softness and poor cell adhesion properties, the AdSCs remained in an undifferentiated state, but their multipotency was reduced. These results provide important insights into stem cell delivery systems using IP hydrogels.
... Although fat-derived mesenchymal cells have been studied for several decades, in 2001, a significant finding was reported that a stem cell population of mesenchyme origin exists in lipoaspirate that could be isolated and maintained in-vitro for extended periods (Zuk et al., 2001;Zuk et al., 2002). Furthermore, a study in the following year demonstrated their ability to differentiate into neural-like cells (Safford et al., 2002). Subsequently, their transdifferentiation potential was extended to numerous other cell lineages. ...
Article
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Adipose tissue-derived stromal vascular fraction (AdSVF) comprises a heterogeneous cell population, including the multipotent mesenchymal stem cells, hematopoietic stem cells, immune cells, endothelial cells, fibroblasts, and pericytes. As such, multipotent adipose tissue-derived mesenchymal stem cells (AdMSCs), are one of the important components of AdSVF. Commonly used techniques to harvest AdSVF involve enzymatic or non-enzymatic methods. The enzymatic method is considered to be the gold standard technique due to its higher yield. The cellular components of AdSVF can be resuspended in normal saline, platelet-rich plasma, or phosphate-buffered saline to produce a ready-to-use solution. Freshly isolated AdSVF has exhibited promising osteogenic and vasculogenic capacity. AdSVF has already been proven to possess therapeutic potential for osteoarthritis management. It is also an attractive therapeutic option for enhancing wound healing. In addition, the combined use of AdSVF and platelet-rich plasma has an additive stimulatory effect in accelerating wound healing and can be considered an alternative to AdMSC treatment. It is also widely used for managing various orthopaedic conditions in clinical settings and has the potential for regenerating bone, cartilage, and tendons. Autologous AdSVF cells are used along with bone substitutes and other biological factors as an alternative to conventional bone grafting techniques owing to their promising osteogenic and vasculogenic capacity. It can also be used for treating osteonecrosis, meniscus tear, chondromalacia, and tendon injuries in veterinary practice. It has several advantages over in vitro expanded AdMSC, including precluding the need for culturing, reduced risk of cell contamination, and cost-effectiveness, making it ideal for clinical use.
... Although mesenchymal stem cells are found in various tissues; bone marrow and adipose tissue are the most researched tissues [8,32]. Stem cells harvested from adipose tissue have shown the ability of differentiation in a similar way to their bone marrow analogues [33][34][35][36]. Uysal et. ...
Article
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Background: The presence of chronic renal disease(CRD) concurrently with diabetes mellitus(DM) increases the flap failure. Adipose derived stromal vascular fraction (SVF) is known to enhance skin flap viability in both healthy and diabetic individuals. The aim of this experimental study was to investigate the effect of SVF on skin flap viability in rats with DM and CRD. Methods: 48 Sprague-Dawley rats were separated into four groups as follows: group I (control), group II (diabetes mellitus), group III (chronic renal disease), and group IV (diabetes with chronic renal disease).Two dorsal flaps were elevated. Flaps on left side of all groups received 0.5 cc of SVF, while same amount of plasma-buffered saline (PBS) was injected into right side. On postoperative day 7, flaps were harvested for macroscopic, histopathologic and biochemical assessments. Areas of flap survival were measured macroscopically. Blood level of vascular endothelial growth factor (VEGF) was measured after injection of SVF. Results: Macroscopically, SVF has significantly improved flap viability (p < 0.05). Flap viability percentage was lower in DM and CRD groups when compared with healthy control group. In respect of new capillary formation, there was a statistically significant difference between SVF injected flaps and PBS injected sides (p < 0.05). Similarly, VEGF levels were higher in all study groups and there was a significant difference in comparison to control group (p < 0.05). Conclusions: The study showed that injection of SVF increased flap viability via endothelial differentiation and neovascularization. In vivo function of stem cells might be impaired due to uremia and diabetes-related microenviromental changes.
... The presence of mesenchymal stem cells in adipose tissue was first described in 2001 by Zuk et al. [1]. These cells have been characterized by strong regenerative properties and a multilineage differentiation potential comparable to bone marrow mesenchymal stem cells [1][2][3][4]. Since then, the regenerative capacity of adipose tissue along with its antiinflammatory and immunomodulatory properties have been a constant field of research and clinical application. From pure macrofat and nanofat grafting till the enzymatically isolated adipose derived stromal vascular fraction (SVF) and cultured stem cells, the adipose tissue has been studied in a wide range of degenerative diseases [5]. ...
Article
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Adipose tissue and its regenerative products which are isolated with enzymatic or mechanical processing of the harvested fat have been studied in a wide range of degenerative diseases, including osteoarthritis of the knee and hip. Intra-articular injection of these products can provide symptomatic relief of pain and postpone surgery. However, their use in the treatment of thumb carpometacarpal joint (CMCJ) osteoarthritis is limited and just a few studies have been published on that topic. For this reason, a review of the literature was performed by a thorough search of eight terms using the Pubmed database. In total, seven human studies met the selection criteria, including case-control studies, case-series and one case report. In all studies, intra-articular injection of autologous fat in osteoarthritic thumb CMCJ provided reduction in pain and improvement in hand function. Grip and pinch strength showed variable results, from no change to significant improvement. Fat-processing techniques were based on centrifugation and mechanical homogenization but biological characterization of the injected cells was not performed in any study. Although the results are encouraging, a uniformly standardized method of fat processing and the conduction of randomized controlled trials in the future could better evaluate the effectiveness of this procedure for thumb CMCJ osteoarthritis.
... First, we examined the cell morphological changes and toxicity of neural-induced hBM-MSCs after exposure to various PEMF intensities. Previous studies have comprehensively characterized the effects of neural differentiation media on hBM-MSCs [19][20][21][22]. To determine the effect of PEMF on hBM-MSCs and neural induction, we used a basic neural differentiation medium with forskolin and insulin. ...
Article
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Stroke is among the leading causes of death worldwide, and stroke patients are more likely to live with permanent disabilities even after treatment. Several treatments are being developed to improve the quality of life of patients; however, these treatments still have important limitations. Our study thus sought to evaluate the neural differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) at various pulsed electromagnetic field (PEMF) frequencies. Furthermore, the effects of selected frequencies in vivo were also evaluated using a mouse ischemia stroke model. Cell proliferation decreased by 20% in the PEMF group, as demonstrated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and lactate dehydrogenase (LDH) secretion increased by approximately 10% in an LDH release assay. Fluorescence-activated cell sorting (FACS) analysis demonstrated that CD73 and CD105 were downregulated in the PEMF group at 60 Hz. Moreover, microtubule-associated protein 2 (MAP-2) and neurofilament light chain (NF-L) were upregulated in cell cultures at 60 and 75 Hz. To assess the effects of PEMF in vivo, cerebral ischemia mice were exposed to a PEMF at 60 Hz. Neural-related proteins were significantly upregulated in the PEMF groups compared with the control and cell group. Upon conducting rotarod tests, the cell/PEMF group exhibited significant differences in motor coordination at 13 days post-treatment when compared with the control and stem-cell-treated group. Furthermore, the cell and cell/PEMF group exhibited a significant reduction in the expression of matrix metalloproteinase-9 (MMP-9), tumor necrosis factor-alpha (TNF-and interferon-gamma (IFN-γ) in the induced ischemic area compared with the control. Collectively, our findings demonstrated that PEMFs at 60 and 75 Hz could stimulate hBM-MSCs neural differentiation in vitro, in addition to promoting neurogenesis to enhance the functional recovery process by reducing the post-stroke inflammatory reaction.
... Our experimental results showed that iron-overload leads to irreversible damage to neuron cell membranes, followed by increased iron-catalyzed NOS activity, which eventually leads to neuron apoptosis. ADSCs transplantation therapy is a reported promising therapeutic strategy for ICH [18]. Recently, ADSCs were reported to protect neurons against a variety of harmful stimuli [19]. ...
Article
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Background Ferrous ion, a degradation product of hematomas, induces inflammatory reactions and other secondary injuries after intracerebral hemorrhage (ICH). Our study aimed to investigate the specific neuroprotective mechanism of adipose‐derived stem cells (ADSCs) on ferrous ion‐induced neural injury in vitro. Methods ADSCs were co‐cultured with primary cortical neurons in a transwell system treated with ferrous sulfate to generate an in vitro ICH model. ADSCs and cortical neurons were cultured in the upper and lower chambers, respectively. Neuron apoptosis was determined by flow cytometry. The levels of insulin‐like growth factor‐1 (IGF‐1), malondialdehyde (MDA) and nitric oxide synthase (NOS) activity in neuron culture medium were detected with commercial kits. In neurons, protein expression in phosphatidylinositol‐3‐kinase (PI3K)/protein kinase B (Akt) signaling pathway, nuclear factor erythroid 2‐related factor 2 (Nrf2)/heme oxygenase‐1 (HO‐1) signaling pathway and apoptosis‐related proteins were detected by western blot. Results ADSCs attenuated neural apoptosis, reduced MDA levels and NOS activity induced by ferrous sulfate. In neurons, IGF‐1 was increased, as were p‐PI3K, p‐Akt, Nrf2, HO‐1, and Bcl‐2 while cleaved caspase 3 was down‐regulated. Conclusions ADSCs exert neuroprotective effects against ferrous iron‐induced neuronal damage by secreting IGF‐1 and increasing the levels of Akt‐dependent Nrf2/ARE signaling pathway.
... In this work,we considered as transdifferentiation the change of adult stromal cells (hADSC) into NSC. Some of the studies describing the obtention of NSC from ADSC use different animal origins incubated with β-mercaptoethanol (BME) [69], valproic acid/insulin [70], isomethylxanthine/ insulin/indomethacin [71], mixtures of bFGF, EGF, PDGF, BDNF and/or RA, or NGF, BDNF, bFGF, dbcAMP, IBMX and indomethacin [72][73][74]. Many of them report the formation of neurons in culture, for short or over prolonged periods, indicating that despite of the neurogenic potential of ADSC, knowledge of NSC status maintenance factors and a better characterization of human transdifferentiated cells are necessary. ...
Article
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Stem cell therapy is an interesting approach for neural repair, once it can improve and increase processes, like angiogenesis, neurogenesis, and synaptic plasticity. In this regard, adult neural stem cells (NSC) are studied for their mechanisms of proliferation, differentiation and functionality in neural repair. Here, we describe novel neural differentiation methods. NSC from adult mouse brains and human adipose-derived stem cells (hADSC) were isolated and characterized regarding their neural differentiation potential based on neural marker expression profiles. For both cell types, their capabilities of differentiating into neuron-, astrocyte- and oligodendrocytes-like cells (NLC, ALC and OLC, respectively) were analyzed. Our methodologies were capable of producing NLC, ALC and OLC from adult murine and human transdifferentiated NSC. NSC showed augmented gene expression of NES, TUJ1, GFAP and PDGFRA/Cnp. Following differentiation induction into NLC, OLC or ALC, specific neural phenotypes were obtained expressing MAP2, GalC/O4 or GFAP with compatible morphologies, respectively. Accordingly, immunostaining for nestin⁺ in NSC, GFAP⁺ in astrocytes and GalC/O4⁺ in oligodendrocytes was detected. Co-cultured NLC and OLC showed excitability in 81.3% of cells and 23.5% of neuron/oligodendrocyte marker expression overlap indicating occurrence of in vitro myelination. We show here that hADSC can be transdifferentiated into NSC and distinct neural phenotypes with the occurrence of neuron myelination in vitro, providing novel strategies for CNS regeneration therapy. Graphical Abstract Superior Part: Schematic organization of obtaining and generating hNSC from hADSC and differentiation processes and phenotypic expression of neuron, astrocyte and oligodendrocyte markers (MAP2, GFAP and O4, respectively) and stem cell marker (NES) of differentiating hNSC 14 days after induction. The nuclear staining in blue corresponds to DAPI. bar = 100 μm. Inferior part: Neural phenotype fates in diverse differentiation media. NES: nestin; GFAP: Glial fibrillary acidic protein. MAP2: Microtubule-associated protein 2. TUJ1: β-III tubulin. PDGFRA: PDGF receptor alpha. Two-way ANOVA with Bonferroni post-test with n = 3. * p < 0.05 and ** p < 0.01: (NSCiM1 NSC induction medium 1) vs differentiation media.
... Other research groups also have successfully isolated pluripotent stem cells from the subcutaneous fats in different experimental animals that have similar biological characteristics. [22][23][24][25] These cells are termed as adipose-derived stem cells (ASCs) and are widely used as "seed cells" in tissue engineering for animal experiments. In this study, we observed that adipose cells at the linea alba of the CV8 acupoint showed a mass-like distribution. ...
Article
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Background:. Shenque (CV8) acupoint is located on the navel and has been therapeutically used for more than 2000 years in Traditional Chinese Medicine (TCM). However, clinical research on the underlying therapeutic molecular mechanisms of the CV8 acupoint lags far behind. This study aimed to study the mechanisms of umbilical acupoint therapy by using stem cells. Methods:. The morphological characteristics of CV8 acupoint were detected under a stereomicroscope using hematoxylin and eosin (H&E) staining. Oil Red, Masson, and immunohistochemical staining on multi-layered slices were used to identify the type of cells at the CV8 acupoint. Cell proliferation was measured by a cell counting kit-8 (CCK-8) method. Flow cytometry and immunohistochemistry were used for cell identification. Induced differentiation was used to compare the differentiation of cells derived from CV8 acupoint and non-acupoint somatic stem cells into other cell types, such as osteogenic, adipogenic, and neural stem cell-like cells. Results:. Morphological observations showed that adipose tissues at the linea alba of the CV8 acupoint in mice had a mass-like distribution. Immunohistochemical staining confirmed the distribution of stem cell antigen-1 (Sca-1) positive cells in the multi-layered slices of CV8 acupoint tissues. Cells isolated from adipose tissues at the CV8 acupoint exhibited high expression of Sca-1 and CD44 and low expression of CD31 and CD34, and these cells possessed osteogenic, adipogenic, and neurogenic stem cell-like cell differentiation ability. The cell proliferation (day 4: 0.5138 ± 0.0111 vs. 0.4107 ± 0.0180, t = 8.447, P = 0.0011; day 5: 0.6890 ± 0.0070 vs. 0.5520 ± 0.0118, t = 17.310, P
... When treating the cultures with neural inducers, which increase the levels of intracellular cAMP, many cells acquired a typical neural cell morphology, associated with an increase of neuron-specific enolase (NSE) and vimentin. These neural cells have been obtained by using only small diameter MSCs cell layers (Zuk et al., 2002;Safford et al., 2002;Oswald et al., 2004;Gimble et al., 2008;Kim and Cho, 2013). ...
Article
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This review aims to offer a vision of the clinical reality of cell therapy today in intensive medicine. For this, it has been carried out a description of the properties, functions, and Mesenchymal Stem Cells (MSCS) sources to subsequently address the evidence in preclinical models and studies clinical trials with whole cells and models attributed to small extracellular vesicles (sEVs), nanoparticles made up of microvesicles secreted by cells with an effect on the extracellular matrix, and their impact as an alternative towards cell-free regenerative medicine. MSCs are cells that enhance the regenerative capacity which can be differentiated typically in different lineages committed as bone, cartilage, and adipose tissue. On the other hand, small extracellular vesicles are structures that participate notoriously and crucially in intercellular communication, which has led to a change in the concept of the functions and the role that these vesicles play in living organisms, in the restoration of damaged tissues and the inflammatory response and immunological. We present the mechanisms that are involved in the applications of MSCS as whole cells and their sEVs in cell therapy and cell-free therapy as an alternative in regenerative medicine. Considering the structural loss that occurs after surgical procedures for cystic and tumoral pathology in periodontitis, as well as the maxillary atrophy that determines the rehabilitation with dental implants, it is imperative to find satisfactory solutions. The opportunity provided by the findings in stem cells is a recent introduction in the field of oral surgery, based on the regenerative potential that these cells possess to restore defects at different levels of the oral cavity. This review aims to discover the real applications that stem cells may have in our treatments shortly.
... Halvorsen reported ECM mineralization and osteoblast gene expression by human ASCs [69] and the chondrogenic potential of ASCs was revealed in vitro and in vivo by Erickson et al. [70]. Moreover, neurogenic differentiation of murine and human ASCs was reported by Safford [71]. Katz et al. [66] analysed the cell surface of and transcriptionally characterized human adipose-derived adherent stromal cells. ...
Article
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Allergic diseases are immune-mediated diseases. Allergies share a common immunopathogenesis, with specific differences according to the specific disease. Mesenchymal stem/stromal cells (MSCs) have been applied to people suffering from allergic and many other diseases. In this review, the immunologic roles of MSCs are systemically reviewed according to disease immunopathogenesis from a clinical viewpoint. MSCs seem to be a promising therapeutic modality not only as symptomatic treatments but also as causative and even preventive treatments for allergic diseases, including atopic dermatitis and chronic urticaria.
... Il a été démontré que ces 22 ASC provenant de lipoaspiration pouvaient, dépendamment des conditions de culture, se différencier vers plusieurs types cellulaires tels que des adipocytes, des ostéoblastes ou des chondrocytes (Zuk et al., 2002). Certains ont même proposé l'obtention de neurones tandis que d'autres proposaient que ces cellules différenciées soient similaires à des neurones (Fujimura et al., 2005;Safford et al., 2002). Ces ASC sont similaires aux cellules souches mésenchymateuses (MSC pour Mesenchymal Stem Cells). ...
Thesis
Il existe deux types de tissus adipeux (TA). Le tissu adipeux blanc stocke les lipides sous forme de triglycérides. Le tissu adipeux brun possède une signature thermogénique via la protéine UCP1 utilisant les lipides pour former de la chaleur. Il existe aussi des adipocytes qui ont des caractéristiques similaires aux adipocytes bruns (adipocytes beiges) au sein du TA blanc. Le TA sécrète également des hormones lui conférant une fonction endocrinienne. Il maintient l’homéostasie énergétique et peut être altéré de différentes façons, ce qui conduit à des dysfonctionnements métaboliques : Une perte importante du TA dans les lipoatrophies est observée lors d’un traitement antirétroviral hautement actif contre le VIH (thérapie HAART). Ceci amène à des modifications métaboliques graves, dues à des niveaux élevés de lipides circulants et à une résistance à l’insuline systémique. Cette thérapie HAART est composée d’inhibiteurs de la protéase du VIH (IPs) ou de la transcriptase inverse (INTI). Les effets inhibiteurs des IPs sur le processus de différenciation adipocytaire blanche sont bien connus. Cependant, les mécanismes spécifiques qui affectent les différents dépôts adipeux humains distinctement ainsi que la différenciation adipocytaire brune le sont moins. Le cancer est une pathologie caractérisée par la prolifération dérégulée de cellules capables de former des métastases. Les cellules tumorales interagissent activement avec leur microenvironnement, notamment avec le TA qui est présent autour de nombreux organes et qui peut favoriser la progression tumorale (tissu adipeux associé au cancer). Le TA promeut la prolifération des cellules cancéreuses par la sécrétion d’adipocytokines. De plus, les cellules tumorales modifient le TA pour tirer leur énergie des lipides ce qui favorise leur expansion et leur dissémination. Nous avons étudié les interactions entre adipocytes et cellules tumorales de sein puisque le TA fait partie intégrante de la glande mammaire. Mon travail de thèse a consisté à identifier de nouveaux mécanismes moléculaires importants pour le développement physiopathologique et/ou l’altération du TA. Nous avons d’abord étudié les effets des IPs sur la perte de l’auto-renouvellement des progéniteurs adipeux (PAs) (1) et sur les modifications métaboliques des adipocytes (2). Nous étudions aussi les interactions entre les cellules de cancer du sein et le microenvironnement adipeux (3). Tout d’abord, les IPs inhibent l’auto-renouvellement des PAs en diminuant IER3 ce qui déstabilise en aval la boucle autocrine de l’Activine A. Les IPs bloquent la différenciation des PAs en adipocytes. La perte de ces deux processus indique que les IPs induisent des lipoatrophies retrouvées au cours de la thérapie HAART. Par la suite, nous observons que les IPs réduisent l’expression des marqueurs thermogéniques dans les adipocytes beiges et bruns par l’inhibition de la transcription d’UCP1. Ils altèrent aussi l’expression des sirtuines, enzymes antivieillissement. L’utilisation d’un activateur de la sirtuine 1 permet de renverser partiellement les effets des IPs sur l’expression d’UCP1. Enfin, nos résultats démontrent que des mammosphères de cancer de sein induisent la protéine UCP1 dans les adipocytes adjacents. L’adrénomedulline produite par les mammosphères participe à ce processus et nous avons pu caractériser son mécanisme d’action. En conclusion, les travaux réalisés pendant ma thèse ont permis de mieux comprendre les mécanismes par lesquels les IPs inhibent l’auto-renouvellement des progéniteurs adipeux ainsi que l’altération de la signature thermogénique via la perte d’UCP1 dans les adipocytes bruns. Les cellules tumorales, quant à elles, induisent l’expression d’UCP1 résultant en une conversion métabolique des adipocytes blancs en adipocytes bruns.
... Adipose-derived mesenchymal stem cells/stromal cells (ADMSCs) have great potential for cell-based therapy and regenerative medicine due to their capability of immunomodulation and in vitro differentiation into various cell lines such as adipocytes, chondrocytes, osteoblasts [1], neuronal cells [2,3] and hepatocytes [4]. Additionally, growth factors, cytokines, exosomes and microvesicles secreted by MSCs also contribute to their regenerative activity in vivo [5][6][7]. ...
Article
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Background We have observed an increased expression of negative markers in some clinical-grade, xeno- and serum-free cultured adipose-derived mesenchymal stem/stromal cell (ADMSC) samples. It gave rise to concern that xeno- and serum-free conditions might have unexpected effects on human ADMSCs. This study aims to test this hypothesis for two xeno- and serum-free media, PowerStem MSC1 media (PS) and StemMACS MSC Expansion Media (SM), that support the in vitro expansion of ADMSCs. Methods We investigated the expression of negative markers in 42 clinical-grade ADMSC samples expanded in PS. Next, we cultured ADMSCs from seven donors in PS and SM and examined their growth and colony-forming ability, surface marker expression, differentiation, cell cycle and senescence, as well as genetic stability of two passages representing an early and late passage for therapeutic MSCs. Results 15 of 42 clinical-grade PS-expanded ADMSC samples showed an increased expression of negative markers ranging from 2.73% to 34.24%, which positively correlated with the age of donors. This rise of negative markers was related to an upregulation of Human Leukocyte Antigen – DR (HLA-DR). In addition, the PS-cultured cells presented decreased growth ability, lower frequencies of cells in S/G2/M phases, and increased ß-galactosidase activity in passage 7 suggesting their senescent feature compared to those grown in SM. Although MSCs of both PS and SM cultures were capable of multilineage differentiation, the PS-cultured cells demonstrated chromosomal abnormalities in passage 7 compared to the normal karyotype of their SM counterparts. Conclusions These findings suggest that the SM media is more suitable for the expansion of therapeutic ADMSCs than PS. The study also hints a change of ADMSC features at more advanced passages and with increased donor’s age. Thus, it emphasizes the necessity to cover these aspects in the quality control of therapeutic MSC products. Graphical abstract
... Adipose tissuederived stem cells can also serve as a source of stem cells that can undergo neural differentiation. Neurospheres form when adipose-derived stem cells are cultured at high density using a neurogenic differentiation medium, which is usually a serumfree medium with the addition of antioxidants, indomethacin, insulin, and isobutylmethylxanthine (Safford et al. 2002(Safford et al. , 2004. ...
Article
Adult stem cells are undifferentiated cells found in many different tissues in the adult human and animal body and are thought to be important for replacing damaged and dead cells during life. Due to their differentiation abilities, they have significant potential for regeneration and consequently therapeutic potential in various medical conditions. Studies on in vitro cultivation of different types of adult stem cells have shown that they have specific requirements for optimal proliferation and stemness maintenance as well as induced differentiation. The main factors affecting the success of stem cell cultivation are the composition of the growth medium, including the presence of serum, temperature, humidity, and contact with other cells and the composition of the atmosphere in which the cells grow. In this chapter, we review the literature and describe our own experience regarding the influence of the presence of fetal bovine serum in the medium and the oxygen concentration in the atmosphere on the stemness maintenance and survival of adult stem cells from various tissue sources such as adipose tissue, muscle, brain, and testicular tissue.
... The stromal vascular fraction (SVF) derived from adipose tissue contains heterogeneous cell populations such as mesenchymal progenitor/stem cells, pre-adipocytes, endothelial cells, pericytes, T cells, and M2 macrophages. SVF-derived mesenchymal progenitor/stem cells, usually referred as ASCs themselves, can be easily expanded in vitro and have the potential to differentiate into multiple lineages, including myogenic, osteogenic, neurogenic, and hematopoietic pathways [133][134][135][136][137]. The angiogenic properties of these cells have been correlated with a strong paracrine activity, secreting an important number of angiogenesis-related cytokines [136]. ...
Article
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Critical limb ischemia (CLI) constitutes the most severe form of peripheral arterial disease (PAD), it is characterized by progressive blockade of arterial vessels, commonly correlated to atherosclerosis. Currently, revascularization strategies (bypass grafting, angioplasty) remain the first option for CLI patients, although less than 45% of them are eligible for surgical intervention mainly due to associated comorbidities. Moreover, patients usually require amputation in the short-term. Angiogenic cell therapy has arisen as a promising alternative for these “no-option” patients, with many studies demonstrating the potential of stem cells to enhance revascularization by promoting vessel formation and blood flow recovery in ischemic tissues. Herein, we provide an overview of studies focused on the use of angiogenic cell therapies in CLI in the last years, from approaches testing different cell types in animal/pre-clinical models of CLI, to the clinical trials currently under evaluation. Furthermore, recent alternatives related to stem cell therapies such as the use of secretomes, exosomes, or even microRNA, will be also described.
... ADSCs are also important therapeutically in the CNS of animal models of TBI and spinal cord injury (SCI) [18,19]. The explanation for this phenomenon may be the transdifferentiation of ADSCs, which induces them to replace injured neurons [20]. However, ADSCs can synthesize and secrete both growth and neurotrophic elements, such as nerve growth factor, brain-derived neurotrophic factor, vascular endothelial growth factor, and hepatocyte growth factor (HGF), all of which are crucial components for neural damage repair [21][22][23][24][25]. ...
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Microglia are considered neurotoxic when activated. Regulating microglial activity during inflammatory stress is an important aspect in treating many central nervous system (CNS) diseases because microglia produce various substances that affect neurons and glial cells. Adipose-derived mesenchymal stem cells (ADSCs) have been shown to have a therapeutic effect following neuronal damage, which is attributed to the fact that ADSCs can differentiate into neurons or produce neurotrophic factors. However, whether ADSCs play a therapeutic role by affecting microglial activity is unclear. In this study, microglia were stimulated with lipopolysaccharide (LPS) to trigger an inflammatory response, and these activated microglia were co-cultured either directly with ADSCs or in ADSCs-conditioned media (ADSCs-CM). ADSCs or ADSCs-CM significantly inhibited not only microglia migration and phagocytosis but also the secretion of proinflammatory factors by microglia. Furthermore, primary hippocampal neurons were cultured with activated microglia in a Transwell co-culture system, and ADSCs or ADSCs-CM were added to investigate whether ADSCs could influence neuronal apoptosis. ADSCs could significantly inhibit neuronal apoptosis in the inflammatory state. The experimental results provided evidence that ADSCs might control microglial activation by producing different substances, indicating that ADSCs can be effective therapeutic strategies for treating diseases associated with microglial inflammation.
... MSCs, a type of adult stem cells, are multipotent in nature, capable of self-renewal and differentiation into lineages such as bone, adipose, cartilage, and fibroblastic lines. While it is controversial, some research groups have elucidated on the ability of these cells to differentiate into non-mesenchymal tissues [1,[26][27][28]. There are reports documenting MSCs differentiating in several skin components including muscle cells, dermal stromal cells, and epithelial cells which could be an encouraging observation for their application in tissue regeneration technology [29][30][31]. ...
Article
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Chronic/non-healing cutaneous wounds pose a debilitating burden on patients and healthcare system. Presently, treatment modalities are rapidly shifting pace from conventional methods to advanced wound care involving cell-based therapies. Mesenchymal stem cells (MSCs) have come across as a prospective option due to its pleiotropic functions viz. non-immunogenicity, multipotency, multi-lineage plasticity and secretion of growth factors, cytokines, microRNAs (miRNA), exosomes, and microvesicles as part of their secretome for assisting wound healing. We outline the therapeutic role played by MSCs and its secretome in suppressing tissue inflammation, causing immunomodulation, aiding angiogenesis and assisting in scar-free wound healing. We further assess the mechanism of action by which MSCs contribute in manifesting tissue repair. The review flows ahead in exploring factors that influence healing behavior including effect of multiple donor sites, donor age and health status, tissue microenvironment, and in vitro expansion capability. Moving ahead, we overview the advancements achieved in extending the lifespan of cells upon implantation, influence of genetic modifications aimed at altering MSC cargo, and evaluating bioengineered matrix-assisted delivery methods toward faster healing in preclinical and clinical models. We also contribute toward highlighting the challenges faced in commercializing cell-based therapies as standard of care treatment regimens. Finally, we strongly advocate and highlight its application as a futuristic technology for revolutionizing tissue regeneration.Graphical abstract
... AD-MSCs were isolated according to a method described by Safford et al (9) . Adipose tissue (100 mg) was obtained from the inguinal fat of SD rats, then was digested in Hank's balanced salt solution containing 0.075% collagenase type I (Gibco, Carlsbad, CA, USA) at 37 °C for 45 min. ...
... The term adipose-MSCs (AD-MSCs) was the original term used to refer to these stem cells based on their potential for multi-lineage specification (McCoy et al. 2008). Despite significant progress in the characterization of cell surface markers for AD-MSCs (Safford et al. 2002), the therapeutic benefit derived from transplantation of AD-MSCs has yet to be demonstrated in animal models of neurological disease such as AD (Chang et al. 2014;Gerth and Thaller 2019). Transplantation of AD-MSCs can improve animal models of neuronal injury, like tremor recovery and motility in combination-transplanted monkeys (Guilak et al. ...
Article
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Alzheimer's disease (AD) is a neuronal disorder with insidious onset and slow progression, leading to growing global concern with huge implications for individuals and society. The occurrence of AD has been increased and has become an important health issue throughout the world. In recent years, the care of more than 35 million patients with AD costs over $ 600 billion per year, it is approximately 1 percent of the global Gross Domestic Product. Currently, the therapeutic approach is not effective for neurological deficits especially after the development of these major neurological disorders. The discovery of the technique called cell-based therapy has shown promising results and made important conclusions beyond AD using the stem cells approach. Here we review recent progress on stem cell-based therapy in the context of AD.
... A maioria dos estudos sobre o processo de diferenciação neural das células ADAS foram baseados, principalmente, em dois trabalhos publicados no ano de 2000, que diferenciaram as células derivadas da medula óssea para tecido neural (Woodbury et al., 2000;Sanchez-Ramos et al., 2008). (Safford et al., 2002;Tholpady et al., 2003;Safford et al., 2004;Krampera et al., 2007 (Kandel et al., 2000). Alguns estudos demonstraram a formação de neurônios maduros e funcionais in vitro a partir de células-tronco embrionárias (Okabe et al., 1996;Miles et al., 2004) e células-tronco adultas neurais (Song et al., 2002;Westerlund et al., 2003). ...
Thesis
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Estudo da integridade genômica durante o processo de diferenciação de células-tronco adultas em células com características neurais TESE DE DOUTORADO ANA PAULA FRANCO LAMBERT Porto Alegre, 2009.
... When exposed to specific growth conditions in vitro, ADSCs can be differentiated toward adipogenic [34], osteogenic [67], chondrogenic [68], myogenic [69], and neurogenic lineage [70]. ...
Article
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Adipose tissue-derived stem cells (ADSCs) are pluripotent mesenchymal stem cells found in relatively high percentages in the adipose tissue and able to self-renew and differentiate into many different types of cells. “Extracellular vesicles (EVs), small membrane vesicular structures released during cell activation, senescence, or apoptosis, act as mediators for long distance communication between cells, transferring their specific bioactive molecules into host target cells”. There is a general consensus on how to define and isolate ADSCs, however, multiple separation and characterization protocols are being used in the present which complicate the results’ integration in a single theory on ADSCs’ and their derived factors’ way of action. Metabolic syndrome and type 2 diabetes mellitus (T2DM) are mainly caused by abnormal adipose tissue size, distribution and metabolism and so ADSCs and their secretory factors such as EVs are currently investigated as therapeutics in these diseases. Moreover, due to their relatively easy isolation and propagation in culture and their differentiation ability, ADSCs are being employed in preclinical studies of implantable devices or prosthetics. This review aims to provide a comprehensive summary of the current knowledge on EVs secreted from ADSCs both as diagnostic biomarkers and therapeutics in diabetes and associated cardiovascular disease, the molecular mechanisms involved, as well as on the use of ADSC differentiation potential in cardiovascular tissue repair and prostheses.
... ADSC differentiate into mesodermal lineages such as chondrocytes, osteocytes and adipocytes but are also capable of transdifferentiation to generate neuronal-like cells in vitro (24,25) as well as in vivo (26,27). Furthermore, hADSC are weakly immunogenic due to the absence of MHC-II expression, making these cells suitable for allogeneic stem cell therapies (22,28,29). ...
Thesis
Malgré plus de deux décennies de recherches intensives, les accidents vasculaires cérébraux (AVC) demeurent l'une des principales causes de décès et d'invalidité dans le monde. À ce jour, les seules thérapies admises pour le traitement des AVC ischémiques sont les techniques de reperfusion,restreintes à une très courte fenêtre thérapeutique, puis ensuite la rééducation neurologique. Bien que ces interventions améliorent grandement le devenir des patients, le nombre de personnes éligibles à la reperfusion et la récupération fonctionnelle post-ischémique induite par des exercices de rééducation sont limités. Dans ce contexte de thérapies alternatives limitées, les cellules souches mésenchymateuses (MSC) réduisent de façon significative les déficits fonctionnels dans des modèles expérimentaux d’AVC ischémique, particulièrement celles purifiées du tissu adipeux. Chez l'homme, les MSC améliorent également la récupération fonctionnelle, même si l'effet apparait moins prononcé. Nous proposons donc d'essayer d’augmenter le potentiel thérapeutique de ces cellules MSC en utilisant le pituitary adenylate cyclase-activating polypeptide (PACAP) connu pour ses propriétés protectrices, neurotrophiques et immunomodulatrices dans le cadre de l’ischémie cérébrale. Au cours de cette étude, nous avons développé et évalué le potentiel thérapeutique de MSC purifiées du tissu adipeux (hADSC) et génétiquement modifiées pour exprimer le PACAP, en utilisant un baculovirus recombinant, permettant l’obtention des hADSC-PACAP. Dans un modèle d’occlusion permanente de l’artère cérébrale moyenne, la transplantation différée de hADSC-PACAP améliore, de façon rapide, la récupération fonctionnelle. Celle-ci est associée à une redirection efficace de la réponse inflammatoire microgliale vers un phénotype protecteur de type M2, susceptible de soutenir les mécanismes de réparation tissulaire. En complément de ces observations, une augmentation de la survie à long terme des neuroblastes et une réorganisation précoce des connexions corticales interhémisphériques est aussi observée. Parallèlement à la réduction globale des connexions interhémisphériques vers le cortex controlésionnel induite par la transplantation des hADSC-PACAP, une augmentation sélective de projections interhémisphériques provenant de la zone périlésionnelle et allant vers le cortex somatosensoriel dédié aux vibrisses, situé dans l’hémisphère controlésionnel, a été constatée. Ainsi, malgré l'absence de différences significatives entre les hADSC et les hADSC-PACAP, ces dernières en modulant la réponse inflammatoire vers un phénotype de type M2c/M2d renforcent la récupération fonctionnelle post-ischémique en créant un environnement propice aux mécanismes de réparation tissulaire et de neuroplasticité. Au regard de nos résultats, la transplantation de ces cellules semble favoriser l’établissement ou le réarrangement des connexions neuronales entre certaines aires cérébrales au détriment d’autres régions.
... 14 Our present findings support that ASCs might improve incontinence in patients with SUI by restoring the function of the urethral sphincter. ASCs differentiate into a mesenchymal lineage that includes nerve tissue and vascular endothelial cells 26,27 and might induce differentiation at the level of the cell and its microenvironment by secreting several cytokines involved in angiogenesis. 28 We showed that ADRCs isolated by the Celution system secreted several angiogenesis-related cytokines, including VEGFA, angiopoietin-2, IL-8, HGF, PGF and G-CSF. ...
Article
Objective To assess the characteristics of adipose‐derived regenerative cells, and provide supportive data explaining the mechanism of efficacy observed for the use of these cells in the treatment of stress urinary incontinence. Methods Adipose tissues were harvested by abdominal liposuction from healthy donors and patients with stress urinary incontinence. Adipose‐derived regenerative cells were isolated from tissues using the Celution system, and assessed for their characteristics and ability to differentiate into smooth muscle cells. Results Adipose‐derived regenerative cells isolated by the Celution system developed into fibroblastic colonies. Flow cytometric analysis of adipose‐derived stem cell markers showed that adipose‐derived regenerative cells were positive for CD34 and CD44, and negative for CD31. Immunofluorescence staining after differentiation showed that colony‐forming cells were positive for alpha‐smooth muscle actin, calponin and desmin, which are smooth muscle cell markers. A cytokine release assay showed that adherent cells secreted cytokines associated with angiogenesis, including vascular endothelial growth factor‐A, angiopoietin‐2 and placental growth factor. Conclusions Adipose‐derived regenerative cells collected by the Celution system might have clonogenic capacity and an angiogenetic function. These properties might contribute to the mechanisms through which regenerative cell therapy by periurethral injection of autologous adipose‐derived regenerative cells ameliorates stress urinary incontinence.
... Since the first description of hMSCs isolated from bone marrow [18-34], many pre-clinical and clinical researchers isolated and characterized MSCs from various tissues, such as umbilical cord blood [24,26,[35][36][37][38][39][40][41][42][43][44], adipose tissue [24,26,[45][46][47][48][49][50][51][52][53][54], Wharton's jelly [55][56][57][58][59][60][61][62], amniotic fluid [63][64][65], dental tissue [12,13,[66][67][68][69][70][71][72][73][74], skin and foreskin [75,76], placenta [36,77], salivary gland [78,79], synovial fluid [80,81], synovial tissue [10,11,82,83], endometrium [84,85], limb bud [86], peripheral blood [87][88][89][90], and nasal polyps [91][92][93][94] (Figure 1 and Table 1). ...
Article
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Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated from various tissues in the adult body. MSCs should be characterized by three criteria for regenerative medicine. MSCs must (1) adhere to plastic surfaces, (2) express specific surface antigens, and (3) differentiate into mesodermal lineages, including chondrocytes, osteoblasts, and adipocytes, in vitro. Interestingly, MSCs have immunomodulatory features and secrete trophic factors and immune receptors that regulate the microenvironment in host tissue. These specific and unique therapeutic properties make MSCs ideal as therapeutic agents in vivo. Specifically, pre-clinical and clinical investigators generated inflammatory and fibrotic diseases models, and then transplantation of MSCs into diseases models for therapeutic effects investigation. In this review, we characterize MSCs from various tissues and describe their applications for treating various inflammation and fibrotic diseases.
... Adipose tissue-derived stem cells (ASCs) are multipotent cells that can differentiate into neurogenic linage cells, being isolated from the subcutaneous adipose tissues in humans [1], dogs [2], mice [3] and rats [4,5]. ASCs have a great advantage over stem cells derived from other tissues in that they can be prepared in large amounts with less injury to their donors by liposuction [6]. ...
Article
Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells through nitric oxide (NO) signaling due to up-regulated inducible NO synthase (iNOS) as early as within 3 days. Here, we investigated mechanisms of VPA-promoted neuronal differentiation of ASCs concerning the NO-citrulline cycle, the metabolic cycle producing NO. Cultured rat ASCs were differentiated to mature neuronal cells rich in dendrites and expressing a neuronal marker by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium for 2 h. Inhibitor (α-methyl-d, l-aspartic acid, MDLA) of arginosuccinate synthase (ASS), a key enzyme of the NO-citrulline cycle, abolishes intracellular NO increase and VPA-promoted neuronal differentiation in ASCs. l-Arginine, the substrate of iNOS, restores the promotion effect of VPA, being against MDLA. Immunocytochemistry showed that ASS and iNOS were increased in ASCs expressing neurofilament medium polypeptide (NeFM), a neuronal marker, by VPA and NIM synergistically. Real-time RT-PCR analysis showed that mRNAs of Ass and arginosuccinate lyase (Asl) in the NO-citrulline cycle were increased by VPA. Chromatin immunoprecipitation assay indicated that Ass and Asl were up-regulated by VPA through the acetylation of their associated histone. From these results, it was considered that VPA up-regulated the whole NO-citrulline cycle, which enabled continuous NO production by iNOS in large amounts for potent iNOS-NO signaling to promote neuronal differentiation of ASCs. This may also indicate a mechanism enabling short-lived NO to function conveniently as a potent signaling molecule that can disappear quickly after its role.
... They can give rise to vascular cells, both smoothmuscle cells and endothelial cells [63][64][65]. They have also been shown to be able to differentiate into cells of ectodermic leaflet such as neuronal cells [39,[66][67][68]. Their neurotrophic and angiogenic properties were reported to be due to the secretion of nerve growth factor (NGF), brain-derived 3 Stem Cells International neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GNDF), vascular endothelial growth factor-A (VEGF-A), and angiopoietin-1 [69]. ...
Article
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In the context of bio-interactive dressings used for enhancing wound healing, the use of stromal vascular fraction (SVF) or adipose-derived stem cells (ASCs) hereof derived has not fully exploited yet. Non-cultured SVF, a heterogeneous mesenchymal population of cells, is attractive in the field of dermal regeneration because it can be instantaneously obtained, avoids genomic alterations and is comparatively safer than cultured ASCs. Integra® Dermal Regeneration Template (DRT) was sprinkled with ASCs in complete medium supplemented with 10% Fetal Bovine Serum, or SVF, obtained from emulsified or non-emulsified fat, in medium supplemented with 2% platelet-rich plasma (PRP). The presence and differentiation of cells were evaluated by standard histochemistry and immunohistochemistry, while conditioned media were analysed for VEGF by ELISA. In vitro experiments were conducted to analyse ASC proliferation in the presence of either FBS or PRP. Deposition of ASCs in medium supplemented with FBS caused their integration into Integra® DRT as early as 1 h. ASCs were found as aggregates until 6-10 days without forming organised structures. When seeded onto Integra® DRT, SVF cells in medium supplemented with PRP formed aggregates at early times, which at 7 and 10 days organized into vascular-like structures, lined by CD31- and smooth muscle actin-positive cells. With non-emulsified fat, the lacunar structures did not show an organized distribution of SVF cells. PRP induced ASC proliferation although at lower level than FBS. VEGF secretion was enhanced when fat emulsification was introduced into the protocol. In conclusion, the combination of SVF cells obtained from emulsified fat, PRP and Integra® DRT exhibit synergistic effect on the formation of vessel-like structures indicating a step forward aimed at regenerative surgery for chronic wound healing.
... Apart from being first identified and isolated from bone marrow, MSCs have been also isolated from other sources like adipose tissue, umbilical cord, placenta and fetal membrane, dental pulp, skeletal muscle, amniotic fluid, fetal blood, peripheral blood, Wharton's Jelly and corneal limbus and have been shown to have similar characteristics (Ab Kadir et al. 2012;Campagnoli et al. 2001;Erices et al. 2000;Gronthos et al. 2000;In't Anker et al. 2003;Polisetty et al. 2008;Raynaud et al. 2012;Wang et al. 2004;Zuk et al. 2001). In addition to mesodermal lineage, MSCs have also exhibited transdifferentiation potential into neuroectodermal lineages like neuronal cells and endodermal lineages like hepatocytes and pancreocytes Anghileri et al. 2008;Datta et al. 2011;Gabr et al. 2013;Govindasamy et al. 2011;Hang et al. 2014;Lee et al. 2004;Naghdi et al. 2009;Pavlova et al. 2012;Safford et al. 2002;Stock et al. 2014;Tang et al. 2012). Having regenerative potential and affinity to home to the damaged sites, MSCs have paved way in research and clinical applications in tissue regeneration, bone disorders, metabolic diseases, etc. (Horwitz et al. 2002;Koc et al. 2002;Undale et al. 2009) Other than MSC characteristics like self-renewal, multipotency and regeneration, another characteristic that has drawn the attention of clinicians and researchers is the immunoregulatory aspect of MSCs. ...
Article
Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent progenitor cells of mesodermal origin possessing multilineage differentiation potential and ease of expansion in vitro. Over the years, these cells have gained attention owing to their potential in cell-based therapies in treating various diseases. In particular, the wide spectrum of immunoregulatory/immunomodulatory role of MSCs in various clinical conditions has gained immense attention. The immunomodulatory properties of BM-MSCs are mediated by either cell–cell contact (interactions with various immune cells in a context-dependent manner), paracrine mode of action or extracellular vesicles, making them a potential option as immunosuppressants/immunomodulators in treating various clinical conditions. A plethora of studies have demonstrated that MSCs do so by exhibiting a profound effect on various immune cells for example they can inhibit the proliferation of T cells, B cells, and natural killer cells; modulate the activities of dendritic cells and induce regulatory T cells both in vitro and in vivo. In this review we aim at briefly elucidating the characteristics of BM-MSCs, specifically addressing the current understanding on the hypoimmunogeneticity and immunomodulatory properties of the same with specific reference to their interactions with B cells, T cells, Dendritic cells and natural killer cells. We also aim at reviewing the secretory profile and their role in some clinical conditions that have shown promising outcomes.
... Several chemicals have been cited in the literature and included as parts of chemical cocktails that reportedly cause neural induction, which may include small-molecule chemicals, growth factors, hormones or other proteins, [23][24][25][26][27] such as those containing indomethacin, insulin and isobutylmethylxanthine (IBMX) [28]; and those containing brain-derived neurotrophic factor (BDNF) and retinoic acid [29]. The previous use of small-molecule chemicals such as β-mercaptoethanol (BME), butylated hydroxyanisole (BHA), valproic acid (VA) and dimethyl-sulfoxide (DMSO) produced differentiated cells within a far shorter time than conventional methods, sometimes within minutes or hours [23]. ...
Article
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Stem cells are highly important in biology due to their unique innate ability to self-renew and differentiate into other specialised cells. In a neurological context, treating major injuries such as traumatic brain injury, spinal cord injury and stroke is a strong basis for research in this area. Mesenchymal stem cells (MSC) are a strong candidate because of their accessibility, compatibility if autologous, high yield and multipotency with a potential to generate neural cells. With the use of small-molecule chemicals, the neural induction of stem cells may occur within minutes or hours. Isobutylmethyl xanthine (IBMX) has been widely used in cocktails to induce neural differentiation. However, the key molecular mechanisms it instigates in the process are largely unknown. In this study we showed that IBMX-treated mesenchymal stem cells induced differentiation within 24 h with the unique expression of several key proteins such as Adapter protein crk, hypoxanthine-guanine phosphoribosyltransferase, DNA topoisomerase 2-beta and Cell division protein kinase 5 (CDK5), vital in linking signalling pathways. Furthermore, the increased expression of basic fibroblast growth factor in treated cells promotes phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) cascades and GTPase–Hras interactions. Bioinformatic and pathway analyses revealed upregulation in expression and an increase in the number of proteins with biological ontologies related to neural development and substructure formation. These findings enhance the understanding of the utility of IBMX in MSC neural differentiation and its involvement in neurite substructure development.
... Mesenchymal stem cells are one of the multipotent adult stem cells that can proliferate into a variety of cell types in culture. [1][2][3][4] In clinical practice, various cell therapies have been utilized for damaged tissue regeneration in several diseases, commonly using mesenchymal stem cells obtained from bone marrow. On the contrary, bone marrow as a tissue source has many limitations, such as the amount of bone marrow that can be collected from a donor is limited, the percentage of mesenchymal stem cells contained in bone marrow is not high, and it is necessary to expand culture to a required amount to be administered as regenerative medicine. ...
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In a novel regenerative cell-based treatment developed by us for the patients with stress urinary incontinence, autologous adipose-derived stem cells (ASCs) are injected into the periurethral region and the external urethral sphincter. Since the candidates for this treatment included prostate cancer patients after radical prostatectomy, we investigated the effects of ASCs on prostate cancer cell proliferation in vitro and in vivo to confirm the feasibility of our therapeutic approach. The LNCaP (human prostate cancer cell line) cells and ASCs were co-cultured, and prostate-specific antigen (PSA) concentration in their culture medium supernatant was measured at 48 and 96 h. The PSA concentration significantly decreased in the coculture medium supernatant as compared to the culture medium with LNCaP cells alone. On the contrary, PSA concentrations in the culture medium of LNCaP cells were not affected by supplementation with ASC culture supernatant. After subcutaneous transplantation of LNCaP cells, with or without ASCs, in immunodeficient mice, tumor growth was compared. The growth of LNCaP xenograft tumor in immunodeficient mice was significantly suppressed by ASC addition. These results indicated that ASCs inhibit prostate cancer cell growth, without no proliferative effect on prostate cancer cells.
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Background: Ovarian transplantation has unique advantages in the preservation of female fertility, especially in young women with cancer who need chemotherapy. However, a large number of follicles are lost because of ischemia during ovarian tissue transplantation. While it has been reported that adipose-derived mesenchymal stem cells (ADSCs) accelerate angiogenesis, the transplanted ADSCs usually diffuse quickly from the target tissue. Urinary bladder matrix (UBM) is an extracellular matrix biomaterial that has a complete basement membrane and provides a foundation for transplanted cells to anchor, migrate, and function. In this study, ADSCs on UBM scaffolds (UBM/ADSCs) were transplanted during ovary autotransplantation in rats to test whether collagen/ADSCs have a better therapeutic effect than transplantation of ADSCs alone. Method: A total of 30 rats were divided into 5 groups of 6 rats in each . untreated-control, oophorectomy , autograft , autograft + ADSCs (ADSC) and autograft + UBM/ADSCs(UBM/ADSC). 28 days after ovary autografting, follicle number, serum concentrations of follicular stimulating hormone and anti-Mullerian hormone and apoptosis rate were also estimated. At 7and 28 days post ovary autografting, angiogenesis was detected. The estrous cycle recovery was measured.The results were analyzed using one-way analysis of variance (ANOVA) and Tukey test, and the means were significantly different at P< 0.05. Results: The number of both growing follicles and primordial follicles in rats in the ADSC/UBM and ADSC groups was significantly higher than that in rats in the autograft group (P <0.05). Follicle stimulating hormone levels in rats in the ADSC/UBM group were significantly decreased and anti-Müllerian hormone levels increased compared to control rats (P <0.05). Apoptosis rate in the UBM/ ADSC group was lower than the autograft group (P<0.05). The angiogenesis was accelerated following ADSC/UBM transplantation.Rats in the ADSC/UBM and ADSC groups showed better estrous cycle recovery than rats in the autograft group (P<0.05). Conclusions: UBM increases the retention of ADSCs in ovaries and contributes to long-term restoration of ovarian function. UBM/ADSC transplantation may be a promising candidate for ovarian transplantation.
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Lysosomal enzymes help in the degradation of various complex biomolecules. Mutations arising from genes encoding them result in deficiency of these enzymes causing serious disturbances in specific metabolic pathways. Krabbe Disease (KD), is one such condition, where galactocerebrosidase enzyme deficiency occurs which is critical for galactosylceramide (GalCer) degradation, which if present abundantly results in a toxic secondary metabolite psychosine accumulation in myelin. It also incites globoid cell infiltration, ultimately resulting in myelination cessation affecting both central nervous system (CNS) and peripheral nervous system. Current life-supportive treatments are not curative since accompanied limitations impede attaining better therapeutic efficacy. Also, the complex pathogenic cascade makes addressing the target difficult. Therapeutic efficacy can be improved by designing ideal drug delivery systems with potential therapeutic agents. This chapter discusses current treatment modalities followed in KD and challenges associated along with a few novel therapeutic approaches which might provide leads for treatment improvements and advancements.
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Mammalian nervous system is the most complex and little understood system of the body. An ailment to any structure of the central nervous system often leads to the mal- or non-function of the particular body organ or system controlled centrally. Currently, no therapeutic modality addresses these ailments in an effective way. As such regenerative medicine employing stem cells are being evaluated. Central nervous system is not devoid of stem cells; however, the limited concentration and their specific localization together with the environmental effect make their actions little effective. As such mesenchymal stem cells (MSCs) are being evaluated due to their characteristic properties like multiplication, differentiation, homing, immunomodulation/anti-inflammatory, antioxidant and reduction of apoptosis. In humans, more extensive studies are conducted as compared to the animals. There are numerous studies demonstrating MSCs in vitro neural-like cell differentiation, although some questions remain to be answered. In vivo applications are extended to various affections involving the meningoencephalomyelitis of unknown origin (MUO), canine distemper, global hypoxia injury, myelomeningocele (MMC) Parkinson’s disease (PD), spinal cord injury (SCI), intervertebral disc disease and Wobbler syndrome in animals. In all these studies, MSCs potential therapeutic role occurs through immunomodulation/anti-inflammatory actions (inhibiting astrogliosis and microgliosis), angiogenesis and growth and proliferation of local neural cells. Such a potential is further enhanced with the gene transfection and addition of scaffolds. However, the literature available on MSCs is limited enough to provide evidence-based medicine in case of different neural affections. The current chapter focuses on MSCs properties in relation to the central nervous system and their potential therapeutic applications in different clinical ailments.KeywordsCNSMesenchymal stem cellsWobbler syndromeParkinson’s diseaseSpinal cord injuryIntervertebral disc disease
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Neuronal differentiation of adipose tissue-derived stem cells (ASCs) is potently promoted by valproic acid (VPA) through a gaseous signaling molecule, nitric oxide (NO). Here, we investigated the involvement of hydrogen sulfide (H2S), another gaseous signaling molecule, in neuronal differentiation of ASCs. VPA-promoted neuronal differentiation of ASCs was accompanied by increased intracellular H2S and sulfane sulfur with increased mRNA expression of enzymes synthesizing sulfane sulfur including cystathionine β-synthase (CBS), of which inhibition reduced the differentiation efficiency. H2S donors, GYY4137 (GYY) or NaHS, potently promoted neuronal differentiation of ASCs when cAMP-elevating agents, dibutyryl cyclic adenosine monophosphate and isobutyl methyl-xanthine, were added as neuronal induction medium (NIM). Neuronal differentiation of ASCs promoted by NaHS or GYY was accompanied by Ca²⁺ entry and increased mRNA expression of voltage-gated Ca²⁺ channels. NaHS or GYY also increased mRNA expression of enzymes of the NO-citrulline cycle including inducible NO synthase (iNOS). It was concluded from these results that H2S potently promoted differentiation of ASCs into neuronal cells expressing functional voltage-gated Ca²⁺ channels with the aid of cAMP-elevating agents, involving NO-mediated signaling cascade. These effects of H2S were also considered as a partial mechanism for the VPA-promoted neuronal differentiation of ASCs.
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Introduction: The knowledge in the field of nerve regeneration is incomplete; there is a constant input of information regarding better techniques, materials, and more importantly the use of regenerative medicine. The adipose-derived stem cell is a new tool in this field with promising results and clearly stated advantages, like ease of harvest and availability. Numerous studies have presented viable options regarding the use of the adipose-derived stem cell; one of the more demanding fields is nerve regeneration. One can say that nerve microsurgery has reached its potential and new techniques are necessary in order to obtain better results. The use of fat grafting can be an answer. Material and method: An experimental model was used in order to understand the influence of the fat graft on nerve regeneration. Both sural nerves from 10 rats were transected and microsurgically coapted; on the left side, the sutured nerve was covered by processed fat from the inguinal region. Histology and ultrasound were used in order to quantify the results. Result and discussions: The histology results, as well as the ultrasound study showed a positive influence of the adipose tissue graft in the process of nerve regeneration, especially regarding scar tissue quality and influencing the microenvironment around the nerve coaptation site. Conclusions: Adipose tissue addition around neurorrhaphy has multiple positive effects on this regeneration, starting from its anti-inflammatory effect, stem cell source with differentiation potential for activated Schwann cells, to indirect action on the corresponding muscle.KeywordsPeripheral nerveNerve regenerationStem cellAdipose tissue
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Background Fluoxetine hydrochloride is one of the most commonly used antidepressants in the selective serotonin reuptake inhibitor class. The aim of work: The study was conducted to detect the effect of chronic fluoxetine treatment on pars distalis and the possible therapeutic effect of adipose-derived mesenchymal stem cells (ADSCs). Material and Method Thirty healthy male adult albino rats were classified into four groups. Control group (Group I) included fifteen rats. Fluoxetine treated (Group II) included five rats that received 24 mg/kg/day of fluoxetine dissolved in 1.0 ml of tap water once a day for 30 days. Fluoxetine group treated with ADSCs (Group III) included five rats that received fluoxetine as group (II) for 30 days, then supplied once by ADSCs at a dose of 1×10⁶ cells/rat in the tail vein suspended in 0.5 ml of phosphate-buffered saline (PBS). Recovery group (Group IV) included five rats that received fluoxetine as the group (II) then received no treatment till the end of the experiment. Samples from pars distalis were processed for light, electron microscopic examination, morphometrical and statistical analyses. Results In the fluoxetine-treated group, there was a disruption in cellular architecture, size, shape, and staining characteristics of pars distalis cells. The administration of ADSCs significantly improved the microscopic appearance of cells, while the recovery group showed some histological changes similar to the fluoxetine group. Conclusion Fluoxetine induced various deleterious changes in the pars distalis of albino rats. These changes were almost corrected by the ADSCs treatment.
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The constantly evolving field of regenerative medicine deals with replacing, repairing, or regenerating tissue by using multidisciplinary scientific fields for humans and animals suffering from various injuries to severe diseases. Tissue engineering takes cell biology, materials science, and engineering principles to replace or repair damaged tissues. The scale of stem cell applications in regenerative medicine has increased extensively to develop various clinical-based treatments and potential stem cell-based therapies. This chapter provides an overview of different stem cell sources and their self-renewal, differentiation mechanisms in the regenerative medicine field with the importance of proteomics analyses to understand the stem cell biological processes. Additionally, the importance of biomaterials selection for stem cell-based regenerative medicine and tissue engineering applications is also discussed in this chapter.
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The differentiation of adipose tissue-derived stem cells (ASCs) to neuronal cells is greatly promoted by valproic acid (VPA), and is synergistically enhanced by the following treatment with neuronal induction medium (NIM) containing cAMP-elevating agents. In the present study, we investigated the synergism between VPA and NIM in neuronal differentiation of ASCs, assessed by the expression of neurofilament medium polypeptide (NeFM), with respect to Ca²⁺ entry. VPA (2mM) treatment for 3 days followed by NIM for 2 h synergistically increased the incidence of neuronal cells differentiated from ASCs to an extent more than VPA alone treatment for 6 days, shortening the time required for the differentiation. VPA increased intracellular Ca²⁺ and the mRNAs of voltage-gated Ca²⁺ channels, Cacna1b (Cav2.2) and Cacna1h (Cav3.2), in ASCs. Inward currents through Ca²⁺ channels were evoked electrophysiologically at high voltage potential in ASCs treated with VPA. NIM reduced the mRNAs of NeFM and Cacna1b in VPA-promoted neuronal differentiation of ASCs. It was concluded that functional N-type voltage-gated Ca²⁺ channels (Cav2.2) are selectively expressed in VPA-promoted neuronal differentiation of ASCs. NIM seems to enhance the mRNA translation of molecules required for the differentiation. Neuronal cells obtained from ASCs by this protocol will be used as a cell source for regenerative therapy of neurological disorders associated with altered Cav2.2 activity.
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With an increase in the incidence of neurodegenerative diseases, a need to replace incapable conventional methods has arisen. To overcome this burden, stem cells therapy has emerged as an efficient treatment option. Endeavours to accomplish this have paved the path to neural regeneration through efficient neuronal transdifferentiation. Despite their potential, the use of stem cells still entails several limitations, such as low differentiation efficiency and difficulties in guiding differentiation. The process of neural differentiation through the stem cells is achieved through the use of chemical inducers or growth factors and their direct introduction reduces their bioavailability in the system. To address these limitations, neural regeneration ventures require growth factors to be effectively implemented on stem cells in order to produce functional neuronal precursor cells. An efficient technique to achieve it is through the delivery of growth factors via microcarriers for their sustained release. It ensures the presence of commensurable concentration even at later stages of neuronal transdifferentiation. Nanofibers and nanoparticles, along with liposomes and such, have been used to implement this. The interaction between such carriers and the growth factors is mainly electrostatic. Such interaction enables them to form a stable assembly through immobilisation of the growth factor either onto their surfaces or within the core of their structures. The rate of sustained release depends upon the release kinetics associated with the polymeric structure employed and its interaction with the encapsulated growth factor. The sustained release ensures that the stem cells immerse under the effect of the growth factors for a prolonged period, ultimately aiding in the formation of cells showing ample characteristics of neuron precursors. This review analyses the various carriers that have been employed for the release of growth factors in an orderly fashion and their constituents, along with the advantages and the limitations they pose in delivering the growth factors for facilitating the process of neuronal transdifferentiation. Graphical abstract
Article
Background: Archeological archives report cranioplasty as 1 of the oldest surgical procedures; however, it was not until the last century that true advances have been made. Alternative approaches are necessary to achieve optimal closure of the defect with fewer adverse effects. We aim to evaluate the use of human adipose-derived stem cells (hADSCs) alone or seeded in scaffolds as the main treatment for cranial bone defects and to assess human patient outcomes. Methods: A systematic review was performed by querying PubMed, Ovid MEDLINE, EMBASE, and Cumulative Index to Nursing and Allied Health Literature databases with the MeSH terms: "adipose-derived stem cells," "cranial bone defect," "stromal vascular factor," "fat grafting," as well as synonyms in combinations determined by our search strategy. We included human models that used hADSCs as primary therapy. We excluded studies in languages other than English. Results: One hundred ninety-four studies were identified after removal of duplicates. Four articles that used hADSCs as the main therapy to treat calvarial defects in humans were included. One article applied the cell therapy alone, and 3 used β-tricalcium phosphate granules as a scaffold to seed the hADSCs. Conclusions: Bone regeneration was reached in a short and intermediate period using autologous hADSCs in humans with no major adverse effects in all 4 articles included. A long-term follow-up study (6 years) exhibited late infections and reabsorption of the β-tricalcium phosphate scaffold seeded with hADSCs.
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Adipose-derived mesenchymal stem/stromal cells (ASCs) are an adult stem cell population able to self-renew and differentiate into numerous cell lineages. ASCs provide a promising future for therapeutic angiogenesis due to their ability to promote blood vessel formation. Specifically, their ability to differentiate into endothelial cells (ECs) and pericyte-like cells and to secrete angiogenesis-promoting growth factors and extracellular vesicles (EVs) makes them an ideal option in cell therapy and in regenerative medicine in conditions including tissue ischemia. In recent angiogenesis research, ASCs have often been co-cultured with an endothelial cell (EC) type in order to form mature vessel-like networks in specific culture conditions. In this review, we introduce co-culture systems and co-transplantation studies between ASCs and ECs. In co-cultures, the cells communicate via direct cell-cell contact or via paracrine signaling. Most often, ASCs are found in the perivascular niche lining the vessels, where they stabilize the vascular structures and express common pericyte surface proteins. In co-cultures, ASCs modulate endothelial cells and induce angiogenesis by promoting tube formation, partly via secretion of EVs. In vivo co-transplantation of ASCs and ECs showed improved formation of functional vessels over a single cell type transplantation. Adipose tissue as a cell source for both mesenchymal stem cells and ECs for co-transplantation serves as a prominent option for therapeutic angiogenesis and blood perfusion in vivo.
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Background: We have observed an increased expression of negative markers in some clinical-grade, xeno- and serum-free cultured adipose-derived mesenchymal stem/stromal cell (ADMSC) samples. It gave rise to concern that xeno- and serum-free conditions might have unexpected effects on human ADMSCs. This study aims to test this hypothesis for two xeno- and serum-free media, PowerStem MSC1 media (PS) and StemMACS MSC Expansion Media (SM), that support the in vitro expansion of ADMSCs. Methods: We investigated the expression of negative markers in 42 clinical-grade ADMSC samples expanded in PS. Next, we cultured ADMSCs from seven donors in PS and SM and examined their growth and colony-forming ability, surface marker expression, differentiation, cell cycle and senescence, as well as genetic stability of two passages representing an early and late passage for therapeutic MSCs. Results: 15 of 42 clinical-grade PS-expanded ADMSC samples showed an increased expression of negative markers ranging from 2.73% to 34.24%, which positively correlated with the age of donors. This rise of negative markers was related to an upregulation of Human Leukocyte Antigen – DR (HLA-DR). In addition, the PS-cultured cells presented decreased growth ability, lower frequencies of cells in S/G2/M phases, and increased ß-galactosidase activity in passage 7 suggesting their senescent feature compared to those grown in SM. Although MSCs of both PS and SM cultures were capable of multilineage differentiation, the PS-cultured cells demonstrated chromosomal abnormalities in passage 7 compared to the normal karyotype of their SM counterparts. Conclusions: These findings suggest that the SM media is more suitable for the expansion of therapeutic ADMSCs than PS. The study also hints a change of ADMSC features at more advanced passages and with increased donor’s age. Thus, it emphasizes the necessity to cover these aspects in the quality control of therapeutic MSC products.
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Mesenchymal stem cells (MSCs) are currently available for a range of applications and have become a good material for regenerative medicine, tissue engineering, and disease therapy. MSCs are self-renewing, multipotent progenitor cells with multilineage potential to differentiate into cell types of mesodermal origin, such as adipocytes, osteocytes, and chondrocytes and exert potent immunosuppressive potentials. In the present review, we highlight the currently reported variations in the differentiation potential of MSCs from different tissue sources, the minimal criteria to define MSCs from various tissue environments and provide a detailed description of MSCs surface markers. Furthermore, MSCs immunomodulatory features secrete cytokines and immune receptors which regulate the microenvironment in the host tissue also revisits in detail. We propose that there are likely more sources of MSCs waiting to be discovered. We need to Standardize MSC s characterization by selecting markers for isolation, cellular and molecular mechanisms involved in MSC-mediated immune modulation, and other functionalities of MSCs should be characterized prior to use in clinical applications.
Technical Report
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Standards for Autologous Adipose-Tissue Derived Stem Cells/Stromal Vascular Fraction Cells (ADSCs/SVFCs) Therapy (Version 2)
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Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have an enormous potential; however, their potential clinical application is being arrested due to various limitations such as teratoma formation followed by tumorigenesis, emergent usage, and the quality control of cells, as well as safety issues regarding long-term culture are also delaying their clinical application. In addition, human ES cells have two crucial issues: immunogenicity and ethical issues associated with their clinical application. The efficient generation of human iPS cells requires gene transfer, yet the mechanism underlying pluripotent stem cell induction has not yet been fully elucidated. Otherwise, although human adult regenerative cells including mesenchymal stem cells have a limited capacity for differentiation, they are nevertheless promising candidates for tissue regeneration in a clinical setting. This review highlights the use of regenerative cells for transplantation in hepatic failure.
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Objective The aim of this study is to establish a unilateral tongue atrophy model by cutting the hypoglossal nerve and to evaluate the safety and feasibility of a fat injection of adipose-derived stem cells (ADSCs) to restore swallowing function. Methods A total of 12 rabbits were randomized to three groups; the ADSCs+fat group (n=4), the fat group (n=4) and the control group (n=4). All rabbits were treated with denervation of the left hypoglossal nerve and their conditions including body weight and food intake were checked during follow-up periods (8 weeks). At 4 weeks after the transection of the nerve, rabbits received the injection therapy into the denervated side of the tongue with 1.0mL fat tissue premixed with 0.5mL ADSCs in the ADSCs+fat group, 1.0mL fat tissue premixed with 0.5mL PBS in the fat group and 1.5mL PBS in the control group. Rabbits were euthanized 8 weeks post-treatment and resected tongues were collected, formalin-fixed and paraffin embedded. To evaluate the change of the intrinsic muscles of the tongue, muscle fibers around the treatment area was analyzed by evaluating 5 consecutive hematoxylin-eosin slides per rabbit. Results Food intake did not decrease upon nerve denervation, and none of the rabbits displayed adverse effect such as aspiration, surgical wound dehiscence or infection. No significant body weight changes were found between the three groups at 4 and 8 weeks after nerve transection (p>0.05). In the control group, the denervated side of tongue had significantly smaller muscle fiber areas and diameters compared to the non-denervated side (p<0.05). The ADSCs+fat group demonstrated a larger area of inferior longitudinal muscle fibers compared to the control and the fat groups (582±312µm² vs. 405±220µm² and 413±226µm²; p<0.05). A significant thicker lesser diameter of inferior longitudinal muscle fibers was found in the ADSCs+fat group compared to the control and the fat groups (24±8µm vs. 20±6µm and 20±7µm; p<0.05). Conclusion The rabbit tongue atrophy model was found suitable for the assessment of muscle change after nerve transection. Fat injection therapy with ADSCs demonstrated great potential to prevent the muscle atrophy after denervation and to promote the muscle regeneration around the injection area.
Chapter
Adipose-derived stem cells (ASCs) reside in the stromal compartment of adipose tissue and can be easily harvested in large quantities through a clinically safe liposuction procedure. ASCs do not induce immunogenic reactions and rather exert immunosuppressive effects. Therefore, they can be used for both autologous and allogeneic transplantations. They hold great promise for cell-based therapies and tissue engineering. A prerequisite to the realization of this promise is the development of successful cryopreservation methods for ASCs. In this chapter, we describe a xeno-free- and chemically defined cryopreservation protocol, which can be used for various clinical applications of ASCs.
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The development of freezing and freeze-drying processes for biological samples requires knowledge of the thermophysical properties of the biomaterial and protectant solutions involved. This chapter provides an introduction on the use of differential scanning calorimetry (DSC) to study thermophysical properties of biomaterials in protective solutions. It covers specific methods to study thermal events related to freezing and drying processes including crystallization, eutectic formation, glass transition, devitrification, recrystallization, melting, molecular relaxation, and phase separation.
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Patients with chronic stroke have currently little hope for motor improvement towards regaining independent activities of daily living; stem cell treatments offer a new treatment option and needs to be developed. Patients with chronic stroke (more than 3 months prior to stem cell treatment, mean 21.2 months post‑stroke) were treated with CD271+ stem cells, 7 patients received autologous and 1 allogeneic cells from first degree relative; administration was intravenous in 1 and intrathecal in 7 patients. Each patient received a single treatment consisting of 2‑5x106 cells/kg and they were followed up for up to 12 months. There were significant improvements in expressive aphasia (2/3 patients) spasticity (5/5, of which 2 were transient), and small improvements in motor function (2/8 patients). Although motor improvements were minor in our chronic stroke patients, improvements in aphasia and spasticity were significant and in the context of good safety we are advocating further administration and clinical studies of CD271+ stem cells not only in chronic stroke patients, but also for spastic paresis/plegia; a different, yet unexplored application is pulmonary emphysema
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Neurogenesis in the mammalian central nervous system is believed to end in the period just after birth; in the mouse striatum no new neurons are produced after the first few days after birth. In this study, cells isolated from the striatum of the adult mouse brain were induced to proliferate in vitro by epidermal growth factor. The proliferating cells initially expressed nestin, an intermediate filament found in neuroepithelial stem cells, and subsequently developed the morphology and antigenic properties of neurons and astrocytes. Newly generated cells with neuronal morphology were immunoreactive for gamma-aminobutyric acid and substance P, two neurotransmitters of the adult striatum in vivo. Thus, cells of the adult mouse striatum have the capacity to divide and differentiate into neurons and astrocytes.
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The dentate gyrus of the hippocampus is one of the few areas of the adult brain that undergoes neurogenesis. In the present study, cells capable of proliferation and neurogenesis were isolated and cultured from the adult rat hippocampus. In defined medium containing basic fibroblast growth factor (FGF-2), cells can survive, proliferate, and express neuronal and glial markers. Cells have been maintained in culture for 1 year through multiple passages. These cultured adult cells were labeled in vitro with bromodeoxyuridine and adenovirus expressing beta-galactosidase and were transplanted to the adult rat hippocampus. Surviving cells were evident through 3 months postimplantation with no evidence of tumor formation. Within 2 months postgrafting, labeled cells were found in the dentate gyrus, where they differentiated into neurons only in the intact region of the granule cell layer. Our results indicate that FGF-2 responsive progenitors can be isolated from the adult hippocampus and that these cells retain the capacity to generate mature neurons when grafted into the adult rat brain.
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Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.
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Many organs contain connective tissue or stromal cells and these cells play important roles in growth, development and tissue repair. Subcutaneous adipose tissue represents an accessible reservoir for the isolation of human stromal cells. Ex vivo, the adipose tissue-derived human stromal cells can be expanded more than 100-fold. These primary cultures respond to adipogenic agonists by accumulating lipid and expressing adipocyte specific proteins, including leptin and the peroxisome proliferator-activated receptor gamma (PPARgamma). In contrast, when the adipose tissue-derived stromal cells are exposed to osteogenic factors, they display osteoblastic gene markers and mineralize their extracellular matrix. This work demonstrates that subcutaneous adipose tissue is a readily available source of multipotential stromal cells. It is possible that these cells will be used clinically to treat a broad range of orthopedic, rheumatologic and periodontal disorders.
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Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.
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A battery of monoclonal antibodies (mAbs) against brain cell nuclei has been generated by repeated immunizations. One of these, mAb A60, recognizes a vertebrate nervous system- and neuron-specific nuclear protein that we have named NeuN (Neuronal Nuclei). The expression of NeuN is observed in most neuronal cell types throughout the nervous system of adult mice. However, some major cell types appear devoid of immunoreactivity including cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells. NeuN can also be detected in neurons in primary cerebellar cultures and in retinoic acid-stimulated P19 embryonal carcinoma cells. Immunohistochemically detectable NeuN protein first appears at developmental timepoints which correspond with the withdrawal of the neuron from the cell cycle and/or with the initiation of terminal differentiation of the neuron. NeuN is a soluble nuclear protein, appears as 3 bands (46-48 x 10(3) M(r)) on immunoblots, and binds to DNA in vitro. The mAb crossreacts immunohistochemically with nervous tissue from rats, chicks, humans, and salamanders. This mAb and the protein recognized by it serve as an excellent marker for neurons in the central and peripheral nervous systems in both the embryo and adult, and the protein may be important in the determination of neuronal phenotype.
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Bone marrow contains a population of rare progenitor cells capable of differentiating into bone, cartilage, muscle, tendon, and other connective tissues. These cells, referred to as MSCs, can be purified and culture expanded from animals and humans. This review summarizes recent experimentation focused on characterizing the cellular aspects of osteogenic differentiation, and exploration of the potential for using autologous stem cell therapy to augment bone repair and regeneration. The authors have completed an array of preclinical studies showing the feasibility and efficacy of MSC based implants to heal large osseous defects. After confirming that syngeneic rat MSCs could heal a critical size segmental defect in the femur, it was established that human MSCs form bone of considerable mechanical integrity when implanted in an osseous defect in an immunocompromised animal. Furthermore, bone repair studies in do gs verify that the technology is transferable to large animals, and that the application of this technology to patients at geographically remote sites is feasible. These studies suggest that by combining MSCs with an appropriate delivery vehicle, it may be possible to offer patients new therapeutic options.
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The current study was done to assess if heterogeneity existed in the degree of adipogenesis in stromal cells (preadipocytes) from multiple donors. In addition to conventional lipid-based methods, we have employed a novel signal amplification technology, known as branched DNA, to monitor expression of an adipocyte specific gene product aP2. The fatty acid binding protein aP2 increases during adipocyte differentiation and is induced by thiazolidinediones and other peroxisome proliferator activated receptor γ ligands. The current work examined the adipogenic induction of aP2 mRNA levels in human adipose tissue stromal cells derived from 12 patients (mean age ± SEM, 38.9 ± 3.1) with mild to moderate obesity (mean body mass index ± SEM, 27.8 ± 2.4). Based on branched DNA technology, a rapid and sensitive measure of specific RNAs, the relative aP2 level in adipocytes increased by 679 ± 93-fold (mean ± SEM, n=12) compared to preadipocytes. Normalization of the aP2 mRNA levels to the housekeeping gene, glyceraldehyde phosphate dehydrogenase, did not significantly alter the fold induction in a subset of 4 patients (803.6 ± 197.5 vs 1118.5 ± 308.1). Independent adipocyte differentiation markers were compared between adipocytes and preadipocytes in parallel studies. Leptin secretion increased by up to three-orders of magnitude while measurements of neutral lipid accumulation by Oil Red O and Nile Red staining increased by 8.5-fold and 8.3-fold, respectively. These results indicate that preadipocytes isolated from multiple donors displayed varying degrees of differentiation in response to an optimal adipogenic stimulus in vitro. This work also demonstrates that branched DNA measurement of aP2 is a rapid and sensitive measure of adipogenesis in human stromal cells. The linear range of this assay extends up to three-orders of magnitude and correlates directly with independent measures of cellular differentiation. J. Cell. Biochem. 810:312–319, 2001. © 2001 Wiley-Liss, Inc.
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To understand the mechanism of the sequential restriction of multipotency of stem cells during development, we have established culture conditions that allow the differentiation of neuroepithelial precursor cells from embryonic stem (ES) cells. A highly enriched population of neuroepithelial precursor cells derived from ES cells proliferates in the presence of basic fibroblast growth factor (bFGF). These cells differentiate into both neurons and glia following withdrawal of bFGF. By further differentiating the cells in serum-containing medium, the neurons express a wide variety of neuron-specific genes and generate both excitatory and inhibitory synaptic connections. The expression pattern of position-specific neural markers suggests the presence of a variety of central nervous system (CNS) neuronal cell types. These findings indicate that neuronal precursor cells can be isolated from ES cells and that these cells can efficiently differentiate into functional post-mitotic neurons of diverse CNS structures.
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Bone marrow stromal cells (BMSC) normally give rise to bone, cartilage, and mesenchymal cells. Recently, bone marrow cells have been shown to have the capacity to differentiate into myocytes, hepatocytes, and glial cells. We now demonstrate that human and mouse BMSC can be induced to differentiate into neural cells under experimental cell culture conditions. BMSC cultured in the presence of EGF or BDNF expressed the protein and mRNA for nestin, a marker of neural precursors. These cultures also expressed glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein (NeuN). When labeled human or mouse BMSC were cultured with rat fetal mesencephalic or striatal cells, a small proportion of BMSC-derived cells differentiated into neuron-like cells expressing NeuN and glial cells expressing GFAP.
Article
Neuronal nuclear antigen (NeuN) immunocytochemistry was studied in 15 normal human fetal nervous systems of 8-24 weeks gestation and in four term neonates. Material was derived from products of conception or from autopsy. Antigen retrieval was enhanced for immunocytochemistry by microwave heating of formalin-fixed paraffin sections. NeuN appears highly specific as a marker of neuronal nuclei in human fetal brain. Only rare nuclei are recognized in the germinal matrix. Cerebellar external granule cells are more strongly immunoreactive than postmigratory internal granule cells until 24 weeks gestation; by term most internal and only a few external granule cells are recognized by NeuN antibody. In the cerebrum, some reactive nuclei are demonstrated along radial glial fibers, particularly near the cortical plate. Within the cortical plate, only deep neurons (future layers 4-6) are marked at 19-22 weeks, but by 24 weeks most neurons in the cortical plate exhibit immunoreactivity, though at term some in layer 2 are still non-reactive. Some neurons fail to be recognized by NeuN at all ages: Cajal-Retzius cells, Purkinje cells, inferior olivary and dentate nucleus neurons, and sympathetic ganglion cells are examples. Despite their common origin in the cerebellar tubercle, basal pontine neurons are strongly reactive even before midgestation, hence NeuN does not predict embryonic origin. Neurons of dorsal root and cranial nerve ganglia are reactive even at 8 weeks. This study of normal fetal central nervous system provides a basis for neuropathological evaluation and as a prelude to applications in cerebral dysgeneses.
Article
A battery of monoclonal antibodies (mAbs) against brain cell nuclei has been generated by repeated immunizations. One of these, mAb A60, recognizes a vertebrate nervous system- and neuron-specific nuclear protein that we have named NeuN (Neuronal Nuclei). The expression of NeuN is observed in most neuronal cell types throughout the nervous system of adult mice. However, some major cell types appear devoid of immunoreactivity including cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells. NeuN can also be detected in neurons in primary cerebellar cultures and in retinoic acid-stimulated P19 embryonal carcinoma cells. Immunohistochemically detectable NeuN protein first appears at developmental timepoints which correspond with the withdrawal of the neuron from the cell cycle and/or with the initiation of terminal differentiation of the neuron. NeuN is a soluble nuclear protein, appears as 3 bands (46-48 x 10(3) M(r)) on immunoblots, and binds to DNA in vitro. The mAb crossreacts immunohistochemically with nervous tissue from rats, chicks, humans, and salamanders. This mAb and the protein recognized by it serve as an excellent marker for neurons in the central and peripheral nervous systems in both the embryo and adult, and the protein may be important in the determination of neuronal phenotype.
Article
Cells of neuronal morphology, expressing the 150- and 200-kDa neurofilament proteins, were generated in vitro from populations of neural cells dissociated from adult (greater than 60-day-old) mouse brain. Most of these neurons arose from dividing precursors, as demonstrated by the incorporation of [3H]thymidine during the culture period and autoradiography. Neuronal production was optimal under the conditions in which precursors were initially stimulated with basic fibroblast growth factor and then exposed to medium conditioned by an astrocytic cell line, Ast-1, in serum-free medium. Few, if any, neurons arose in control cultures or in cultures kept in serum and fibroblast growth factor. These results suggest that neuronal precursors exist in the adult mammalian brain, but they require discrete epigenetic signals for their proliferation and differentiation.
Article
Multipotential CNS stem cells receive and implement instructions governing differentiation to diverse neuronal and glial fates. Exploration of the mechanisms generating the many cell types of the brain depends crucially on markers identifying the stem cell state. We describe a gene whose expression distinguishes the stem cells from the more differentiated cells in the neural tube. This gene was named nestin because it is specifically expressed in neuroepithelial stem cells. The predicted amino acid sequence of the nestin gene product shows that nestin defines a distinct sixth class of intermediate filament protein. These observations extend a model in which transitions in intermediate filament gene expression reflect major steps in the pathway of neural differentiation.
Article
This chapter describes the structural properties of neuronal intermediate filaments (IFs) and how these properties influence neuronal-IF assembly dynamics and axonal-transport kinetics. IF proteins, including the neuronal IFs, belong to a complex, multigene family, which has been divided into six types of genes. IF proteins typically contain a central, α-helical rod domain of approximately 310 amino acids, which is subdivided into three coils by short, non-α-helical linker domains of more variable length and sequence. The rod domain is highly conserved in primary and secondary structure among the IFs, particularly at the ends. This rod domain confers a fibrous character upon IF proteins, and by virtue of its hydrophobic heptad repeats yields a hydrophobic strip along one side of the helix to promote dimerization. The amino-terminal head domains of the neuronal IFs exhibit only limited sequence homologies, including an 11-amino acid-stretch highly conserved between rat α-internexin and NF-M. Rich in arginine, they are highly basic, and the prevalence of glycine, proline, and serine residues predicts a structure dominated by β-turns and β-sheets.
Article
The ability to detect infiltration in bone marrow biopsy specimens from patients with disseminated neuroblastoma was assessed by immunohistological and routine histological methods. Frozen cores from 33 staging procedures were tested with UJ13A and UJ127.11. Immunopositive tumour cells were found in 10 of 17 staging procedures in which tumour was detectable by routine histological methods. Positive cells or stromal material were also found in eight of 12 staging procedures in which distorted architecture and fibrosis, but no obvious tumour, had been noted. Paraffin wax embedded cores from 29 of the same staging procedures were tested with antibodies against neurone specific enolase and neurofilament. Only a single core reacted with anti-neurofilament antibody. Neurone specific enolase positive cells or stromal material were found in nine of 15 staging procedures in which obvious tumour was detectable. Although these immunohistochemical techniques proved inferior to routine histology in their ability to detect obvious tumour, the demonstration of immunopositive stromal tissue which was not frankly malignant supports the view that distorted, fibrotic marrow may reflect persistence of neuroectodermal tissue and justifies its distinction from normal marrow when reporting the response to treatment.
Article
This study was designed to correlate cytological features that had previously been established for oligodendrocytes at different developmental stages in vivo and cytological criteria in vitro with the expression of stage-specific cell-surface antigens of cultured oligodendrocytes. Cells obtained from the corpus callosum of 10-day-old C57BL/6J mice were maintained in monolayer cultures and stained with monoclonal antibodies 01 through 012 by indirect immunofluorescence or immunoperoxidase methods. 0 antigen-positive cells were classified according to two criteria: (a) cell shape (type I-III); and (b) cytoplasmic features at the ultrastructural level (class 1-3). Approximately 95% of all 0 antigen-positive cells could be identified as oligodendrocytes by established cytological criteria, thus supporting previous evidence of their glial character from electrophysiological and cell type-specific marker studies. After 12 days in vitro approximately 90% of all morphologically identified oligodendrocytes expressed antigens 03, 04, 05 or 06, which are the first to appear during development in vivo, whereas only 30-40% expressed antigens 011 or 012 which are the last to appear during development in vivo. 01 through 010 antigen-positive oligodendrocytes belong to 3 morphologically distinct cell types: (1) with small (approximately 10 micron in diameter) round cell bodies and few slender processes; (2) with 'hairy eyeball' morphology with a network of processes; and (3) with large, sometimes bipolar cell bodies (up to 30 micron in diameter) surrounded by high amounts of membranous material devoid of cytoplasm. By cytoplasmic criteria at least 90% of all 0 antigen-positive cells fit the description by Mori and Leblond of 'light' to 'medium' oligodendrocytes in vivo, although a clear-cut correlation with expression of early or late appearing 0 antigens was not observed. Typically 'dark' oligodendrocytes were rarely seen in our cultures. 011 and 012 antigen-positive cells are restricted to the group of large oligodendrocytes with high amounts of membranous material, often organized in more or less compact structures (type III). In contrast to the more uniform localization of antigens 01 through 010 over the whole cell surface, antigens 011 and 012 are less strongly detectable on cell bodies than on processes and membranous whirls.
Article
We have isolated and sequenced a cDNA clone encoding the human gamma enolase. Comparison of our cDNA sequence and the rat gamma enolase sequence revealed 97% homology at the level of amino acid sequence. The two coding regions were 91% homologous on the nucleotide level, whereas the 3' noncoding regions were much less homologous (32%). Further comparison of our cDNA sequence with the human alpha enolase revealed an 82% homology at the amino acid level and a 75% homology at the nucleotide level for the two coding regions, whereas the 3' nontranslated regions were only 30% homologous. Using a portion of the 3' nontranslated region of our cDNA, shown to be specific for human gamma enolase, a single 2.5 kb mRNA was detected in human brain tissue. This same gamma enolase message was also found in a number of human normal nonneuronal tissues, and in several human tumor-derived cell lines. Expression of the mRNA for the gamma enolase subunit should thus be used with caution when identifying the cells of neuronal or neuroendocrine origin.
Article
In the human fetal spinal cord at 15 to 16 weeks, glial fibrillary acidic protein (GFAP) was demonstrated within the cytoplasm and processes of cells having the cytological, ultrastructural, and immunocytochemical features of oligodendrocytes--including processes that extend into and contribute to the formation of myelin sheaths. By 17 to 18 weeks, however, GFAP immunoreactivity was no longer evident within such cells. Thus GFAP is expressed by myelin-forming oligodendroglia early in their development.
Article
NNE (non-neuronal alpha-enolase) is a glycolytic enzyme detected in most tissues. NSE (neuron-specific gamma-enolase) is detected in normal neurons and tumors such as neuroblastoma. Staining with antibodies against NSE is therefore used to detect neuroblastoma cells invading bone marrow. Since staining of normal leukocytes has been reported we asked whether bona fide NSE is in fact expressed in normal blood and marrow. We designed nested coding region specific primers for NSE and NNE and, after reverse transcription of mRNA, we amplified the coding region between these primers in a semi-nested polymerase chain reaction. In order to distinguish both iso-mRNAs from each other, we amplified a long (1,047 bp) template in a first round of 30 cycles with primers specific for NNE or NSE. One percent of this product was used in a second round of 30 cycles in which both sense primers and two nested anti-sense primers of alternate specificities yielding shorter products of discernible sizes (768 bp or 619 bp) were added together in the same reaction tube. With this combination of four primers, only that shorter product was amplified to visibility, the specificity of which was homologous to the template produced in the first 30 cycles. Restriction enzyme digestion of the amplified products was used to verify this polymerase chain reaction-based approach for the distinction of isoforms of RNA. This semi-nested polymerase chain reaction clearly allows for the distinction of mRNA for NNE or NSE and shows the presence of transcripts for NSE in normal human leukocytes from blood and bone marrow. This method exploiting short stretches of nucleotide differences in the coding regions for priming can more generally be applied to the distinction of all isoforms of RNA where nested specific primers can be designed. However, the presence of NSE specific transcripts in normal human leukocytes invalidates the use of this highly sensitive method as a disease marker in neuroblastoma.
Article
Marrow stromal cells can be isolated from other cells in marrow by their tendency to adhere to tissue culture plastic. The cells have many of the characteristics of stem cells for tissues that can roughly be defined as mesenchymal, because they can be differentiated in culture into osteoblasts, chondrocytes, adipocytes, and even myoblasts. Therefore, marrow stromal cells present an intriguing model for examining the differentiation of stem cells. Also, they have several characteristics that make them potentially useful for cell and gene therapy.
Article
Marrow stromal fibroblasts (MSFs) are essential for the formation of the haemopoietic microenvironment and bone; however, regulation of MSF proliferation is poorly understood. MSF colony formation was studied in primary mouse and human marrow cell cultures. After a brief exposure to serum, MSF colony formation occurred in the absence of both serum and non-adherent marrow cells, if medium conditioned by marrow cells was present (serum-free conditioned medium, SF-CM). In mouse and human cultures stimulated to proliferate by SF-CM, neutralizing antibodies against PDGF, TGF-beta, bFGF and EGF specifically suppressed MSF colony formation. The degree of supression was species-dependent, with the most profound inhibition achieved in mouse cultures by anti-PDGF, anti-bFGF and anti-EGF, and in human cultures by anti-PDGF and anti-TGF-beta. Serum-free medium not conditioned by marrow cells (SFM) did not support MSF colony formation. In mouse cultures in SFM, human recombinant bFGF and bovine natural bFGF were able to partially substitute for the stimulating effect of SF-CM. Other growth factors, including TGF-beta1, TGF-beta2, PDGF, EGF, IL-6, IGF-I and IGF-II, showed no activity when tested alone. In human cultures in SFM, none of the growth factors, alone or in combination, stimulated MSF colony formation. Mouse and human MSFs grown in SF-CM formed bone and a haemopoietic microenvironment when transplantated into immunodeficient mice in vivo, and therefore were functionally equivalent to MSFs generated in the presence of serum. These data indicate that stimulation of the initial proliferation of an MSF precursor cell is complex, and requires participation of at least four growth factors: PDGF, bFGF, TGF-beta and EGF. In addition, mouse and human MSF precursor cells have different requirements for each of the growth factors.
Article
The in vivo properties of four different neural stem cell lines, generated from embryonic striatum or hippocampus by immortalization with the temperature-sensitive (s) A58/U19 allele of the SV40 Large T-antigen, have been studied with respect to their ability to survive, differentiate, and integrate after transplantation to the adult rat striatum. The cells were labeled with [3H]thymidine prior to grafting, and combined autoradiography and immunohistochemistry was used to characterize their phenotypic differentiation within the adult brain environment. The results show that all four types of cells survived well, up to at least 1.5-6 months postgrafting, without any signs of tissue perturbation or tumor formation. The cells underwent, on average, 2-3 cell divisions during the first 5 days after implantation and exhibited extensive migration over a distance of 1-1.5 mm from the injection site to become morphologically integrated with the surrounding host striatum. The cell number and tissue distribution attained by 2 weeks remained stable for up to 6 months postgrafting with the exception of one cell line, which showed a 40% loss of cells between 2 and 6 weeks. Twice the number of [3H]thymidine-labeled cells were recovered when the cells were grafted into a 1-week-old excitotoxic striatal lesion, probably due to an increased proliferation of the cells in response to the neuron-depleting depleting lesion. The immortalized cells behaved as multipotent neural progenitors. The vast majority of the cells developed a glial-like morphology, 6-14% being clearly GFAP-positive; however, a small but consistent proportion of them (1-3%) expressed MAP-2 and exhibited neuron-like morphology. In mature transplants about 75-80% of the grafted cells were located in the striatal grey matter, and 10-15% in white matter, some of which are proposed to have differentiated into oligodendrocytes. Remaining 5-10% occurred around small blood vessels (resembling pericytes) and in the subventricular zone underneath the ependyma of the lateral ventricle. It is concluded that the ts cell lines are highly suitable for intracerebral transplantation and that they allow the creation of a regionally confined cellular chimeras where the graft-derived glial cells become stably integrated with the resident glial cell matrix.
Article
Progenitor cells were isolated from the developing human central nervous system (CNS), induced to divide using a combination of epidermal growth factor and fibroblast growth factor-2, and then transplanted into the striatum of adult rats with unilateral dopaminergic lesions. Large grafts were found at 2 weeks survival which contained many undifferentiated cells, some of which were migrating into the host striatum. However, by 20 weeks survival, only a thin strip of cells remained at the graft core while a large number of migrating astrocytes labeled with a human-specific antibody could be seen throughout the striatum. Fully differentiated graft-derived neurons, also labeled with a human-specific antibody, were seen close to the transplant site in some animals. A number of these neurons expressed tyrosine hydroxylase and were sufficient to partially ameliorate lesion-induced behavioral deficits in two animals. These results show that expanded populations of human CNS progenitor cells maintained in a proliferative state in culture can migrate and differentiate into both neurons and astrocytes following intracerebral grafting. As such these cells may have potential for development as an alternative source of tissue for neural transplantation in degenerative diseases.
Article
Growth and repair of skeletal muscle are normally mediated by the satellite cells that surround muscle fibers. In regenerating muscle, however, the number of myogenic precursors exceeds that of resident satellite cells, implying migration or recruitment of undifferentiated progenitors from other sources. Transplantation of genetically marked bone marrow into immunodeficient mice revealed that marrow-derived cells migrate into areas of induced muscle degeneration, undergo myogenic differentiation, and participate in the regeneration of the damaged fibers. Genetically modified, marrow-derived myogenic progenitors could potentially be used to target therapeutic genes to muscle tissue, providing an alternative strategy for treatment of muscular dystrophies.
Article
Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34 degrees C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5 degrees C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5 degrees C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10(-8) M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10(-8) M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5 degrees C with ascorbate and beta1-glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.
Article
Mesenchymal stem cells (MSCs) are a population of pluripotent cells within the bone marrow microenvironment defined by their ability to differentiate into cells of the osteogenic, chondrogenic, tendonogenic, adipogenic, and myogenic lineages. We have developed methodologies to isolate and culture-expand MSCs from human bone marrow, and in this study, we examined the MSC's role as a stromal cell precursor capable of supporting hematopoietic differentiation in vitro. We examined the morphology, phenotype, and in vitro function of cultures of MSCs and traditional marrow-derived stromal cells (MDSCs) from the same marrow sample. MSCs are morphologically distinct from MDSC cultures, and flow cytometric analyses show that MSCs are a homogeneous cell population devoid of hematopoietic cells. RT-PCR analysis of cytokine and growth factor mRNA in MSCs and MDSCs revealed a very similar pattern of mRNAs including IL-6, -7, -8, -11, -12, -14, and -15, M-CSF, Flt-3 ligand, and SCF. Steady-state levels of IL-11 and IL-12 mRNA were found to be greater in MSCs. Addition of IL-1alpha induced steady-state levels of G-CSF and GM-CSF mRNA in both cell preparations. In contrast, IL-1alpha induced IL-1alpha and LIF mRNA levels only in MSCs, further emphasizing phenotypic differences between MSCs and MDSCs. In long-term bone marrow culture (LTBMC), MSCs maintained the hematopoietic differentiation of CD34+ hematopoietic progenitor cells. Together, these data suggest that MSCs represent an important cellular component of the bone marrow microenvironment.
Article
Mesenchymal progenitor cells provide a source of cells for the repair of musculoskeletal tissue. However, in vitro models are needed to study the mechanisms of differentiation of progenitor cells. This study demonstrated the successful induction of in vitro chondrogenesis with human bone-marrow-derived osteochondral progenitor cells in a reliable and reproducible culture system. Human bone marrow was removed and fractionated, and adherent cell cultures were established. The cells were then passaged into an aggregate culture system in a serum-free medium. Initially, the cell aggregates contained type-I collagen and neither type-II nor type-X collagen was detected. Type-II collagen was typically detected in the matrix by the fifth day, with the immunoreactivity localized in the region of metachromatic staining. By the fourteenth day, type-II and type-X collagen were detected throughout the cell aggregates, except for an outer region of flattened, perichondrial-like cells in a matrix rich in type-I collagen. Aggrecan and link protein were detected in extracts of the cell aggregates, providing evidence that large aggregating proteoglycans of the type found in cartilaginous tissues had been synthesized by the newly differentiating chondrocytic cells; the small proteoglycans, biglycan and decorin, were also detected in extracts. Immunohistochemical staining with antibodies specific for chondroitin 4-sulfate and keratan sulfate demonstrated a uniform distribution of proteoglycans throughout the extracellular matrix of the cell aggregates. When the bone-marrow-derived cell preparations were passaged in monolayer culture as many as twenty times, with cells allowed to grow to confluence at each passage, the chondrogenic potential of the cells was maintained after each passage.
Article
New neurons are continuously added in specific regions of the adult mammalian central nervous system. These neurons are derived from multipotent stem cells whose identity has been enigmatic. In this work, we present evidence that ependymal cells are neural stem cells. Ependymal cells give rise to a rapidly proliferating cell type that generates neurons that migrate to the olfactory bulb. In response to spinal cord injury, ependymal cell proliferation increases dramatically to generate migratory cells that differentiate to astrocytes and participate in scar formation. These data demonstrate that ependymal cells are neural stem cells and identify a novel process in the response to central nervous system injury.
Article
Stem cells are a valuable resource for treating disease, but limited access to stem cells from tissues such as brain restricts their utility. Here, we injected marrow stromal cells (MSCs) into the lateral ventricle of neonatal mice and asked whether these multipotential mesenchymal progenitors from bone marrow can adopt neural cell fates when exposed to the brain microenvironment. By 12 days postinjection, MSCs migrated throughout the forebrain and cerebellum without disruption to the host brain architecture. Some MSCs within the striatum and the molecular layer of the hippocampus expressed glial fibrillary acidic protein and, therefore, differentiated into mature astrocytes. MSCs also populated neuron rich regions including the Islands of Calleja, the olfactory bulb, and the internal granular layer of the cerebellum. A large number of MSCs also were found within the external granular layer of the cerebellum. In addition, neurofilament positive donor cells were found within the reticular formation of the brain stem, suggesting that MSCs also may have differentiated into neurons. Therefore, MSCs are capable of producing differentiated progeny of a different dermal origin after implantation into neonatal mouse brains. These results suggest that MSCs are potentially useful as vectors for treating a variety of central nervous system disorders.