Stem Cell Reviews and Reports

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Stem cell therapy for cardiac regeneration has been gaining traction as a possible intervention for the reduction of the burden associated with MI and heart failure. However, stem cell therapies have several shortcomings, including poor engraftment, limited improvements in cardiac function, and possible teratogenicity. Recently, extracellular vesicles (EVs) from stem cell sources have been explored as a novel therapy to regenerate the injured myocardium in several animal MI trials. In this systematic review and meta-analysis, we investigate the use of stem cell-derived EVs for cardiac repair preclinical trials in animal MI models. Cochrane Library, Medline, Embase, PubMed, Scopus and Web of Science and grey literature (Canadian Agency for Drugs, Technologies in Health, and Google Scholar) were searched through August 20, 2020 and 37 articles were included in the fnal analysis. The overall efect size observed in EV-treated small animals after MI for ejection fraction (EF) was 10.85 [95 %CI: 8.79, 12.90] and for fractional shortening (FS) was 7.19 [95 %CI: 5.43, 8.96] compared to control-treated animals. The most abundant stem cell source used were mesenchymal stem cells which showed robust improvements in EF and FS (MD = 11.89 [95% CI: 9.44, 14.34] and MD = 6.96 [95% CI: 4.97, 8.96], respectively). Signifcant publication bias was detected for EF and FS outcomes. This study supports the use of EVs derived from stem cells as a novel therapy for cardiac repair after MI. Further investigation in larger animal studies may be necessary before clinical trials. PROSPERO registration number: CRD42019142218.
During the past two decades, pluripotent stem cells have been widely used to study mechanisms of human neural development, disease modeling, and drug discovery in vitro. Especially in the field of Alzheimer's disease (AD), where this treatment is lacking, tremendous effort has been put into the investigation of molecular mechanisms behind this disease using induced pluripotent stem cell-based models. Numerous of these studies have found either novel regulatory mechanisms that could be exploited to develop relevant drugs for AD treatment or have already tested small molecules on in vitro cultures, directly demonstrating their effect on amelioration of AD-associated pathology. This review thus summarizes currently used differentiation strategies of induced pluripotent stem cells towards neuronal and glial cell types and cerebral organoids and their utilization in modeling AD and potential drug discovery.
Endovascular treatment is prevalent as a primary treatment for coronary and peripheral arterial diseases. Although the introduction of drug-eluting stents (DES) dramatically reduced the risk of in-stent restenosis, stent thrombosis persists as an issue. Notwithstanding improvements in newer generation DES, they are yet to address the urgent clinical need to abolish the late stent complications that result from in-stent restenosis and are associated with late thrombus formation. These often lead to acute coronary syndromes with high mortality in coronary artery disease and acute limb ischemia with a high risk of limb amputation in peripheral arterial disease. Recently, a significant amount of research has focused on alternative solutions to improve stent biocompatibility by using tissue engineering. There are two types of tissue engineering endothelialisation methods: in vitro and in vivo. To date, commercially available in vivo endothelialised stents have failed to demonstrate antithrombotic or anti-stenosis efficacy in clinical trials. In contrast, the in vitro endothelialisation methods exhibit the advantage of monitoring cell type and growth prior to implantation, enabling better quality control. The present review discusses tissue-engineered candidate stents constructed by distinct in vitro endothelialisation approaches, with a particular focus on fabrication processes, including cell source selection, stent material composition, stent surface modifications, efficacy and safety evidence from in vitro and in vivo studies, and future directions. Keywords: Endothelial colony forming cells; Endovascular devices; Gene-transferred cells; Human trophoblastic endovascular progenitor cells; Human umbilical vein endothelial cells; In vitro endothelialisation; Mesenchymal stem cells; Stent materials; Stent surface modification; Tissue-engineered stents.
Multipotent stromal cells (MSCs) are widely utilized in therapy for their immunomodulatory properties, but their usage in infectious viral diseases is less explored. This review aimed to collate the current novel use of MSCs in virus-associated conditions, including MSC's susceptibility to virus infection, antiviral properties of MSCs and their effects on cell-based immune response and implementation of MSC therapy in animal models and human clinical trials of viral diseases. Recent discoveries shed lights on MSC's capability in suppressing viral replication and augmenting clearance through enhancement of antiviral immunity. MSC therapy may maintain a crucial balance between aiding pathogen clearance and suppressing hyperactive immune response.
HIV infection continues to be a serious health issue with an alarming global spread, owing to the fact that attempts at developing an effective vaccine or a permanent cure remains futile. So far, the only available treatment for the clinical management of HIV is the combined Anti-Retroviral Therapy (cART), but the long-term cART is associated with metabolic changes, organ damages, and development and transmission of drug resistant HIV strains. Thus, there is a need for the development of one-time curative treatment for HIV infection. The allogeneic transplantation with the Hematopoietic Stem and Progenitor cells (HSPCs) having 32 bp deletion in Chemokine receptor 5 gene (CCR5 Δ32) demonstrated successful HIV remission in the Berlin and London patients, and highlighted that transplantation of CCR5 null HSPCs is a promising approach for a long- term HIV remission. The advent of gene editing technologies offers a new choice of generating ex vivo CCR5 ablated allogeneic or autologous HSPCs for stem cell transplantation into HIV patients. Many groups are attempting CCR5 disruption in HSPCs using various gene-editing strategies. At least two such studies, involving CCR5 gene editing in HSPCs have entered the clinical trials. This review aims to outline the strategies taken for CCR5 gene editing and discuss the challenges associated with the development of CCR5 manipulated HSPCs for the gene therapy of HIV infection.
Cardiac stem cells in adult mouse heart. Hematoxylin and Eosin stained smear of cells obtained after partial enzymatic digestion of adult mouse heart tissue. Cells located on the heart surface were dislodged and collected as a single cell suspension and centrifuged at 1000 g otherwise these stem cells will not be observed as they remain buoyant when spun at 200-300 g. Darkly stained, small sized, spherical stem cells with high nucleo-cytoplasmic ratio were observed interspersed with bigger cardiomyocytes with abundant pink cytoplasm and pale stained nuclei. a. H&E stained section of intact heart surface b-c. H&E stained section of
Characterization of cardiac stem cells. The stem cells expressed OCT-4 [note cells expressing nuclear and cytoplasmic OCT-4]. Smallsized VSELs express nuclear OCT-4 (arrow) whereas slightly bigger CSCs express cytoplasmic OCT-4], SSEA-1, C-KIT and SCA-1. Note cells of variable sizes express C-KIT. Small-sized stem cells expressing C-KIT pellet down only when spun at 1000 g whereas the bigger cells at 200-300 g. Immuno-histochemical localization of C-KIT on the cell
Adult mammalian heart is considered to be one of the least regenerative organs as it is not able to initiate endogenous regeneration in response to injury unlike in lower vertebrates and neonatal mammals. Evidence is now accumulating to suggest normal renewal and replacement of cardiomyocytes occurs even in middle-aged and old individuals. But underlying mechanisms leading to this are not yet clear. Do tissue-resident stem cells exist or somatic cells dedifferentiate leading to regeneration? Lot of attention is currently being focused on epicardium as it is involved in cardiac development, lodges multipotent progenitors and is a source of growth factors. Present study was undertaken to study the presence of stem cells in the pericardium. Intact adult mouse heart was subjected to partial enzymatic digestion to collect the pericardial cells dislodged from the surface. Pericardial cells suspension was processed to enrich the stem cells using our recently published protocol. Two populations of stem cells were successfully enriched from the pericardium of adult mouse heart along with distinct 'cardiospheres' with cytoplasmic continuity (formed by rapid proliferation and incomplete cytokinesis). These included very small embryonic-like stem cells (VSELs) and slightly bigger 'progenitors' cardiac stem cells (CSCs)
In testis, a rare undifferentiated germ cell population with the capacity to regenerate robustly and support spermatogenesis, is defined as spermatogonial progenitor cells (SPCs) population. As a widely used drug for tumor therapy or bone marrow transplantation, busulfan has a severe side effect on SPCs population and causes a consequent infertility. Recently, accumulating evidence revealed the protective role of autophagy in stem cell maintenance under exogenous stress. To better understand the role of autophagy in SPCs fates, we investigated the potential function of autophagy in SPCs under busulfan stress, and found that treatment of busulfan induced the formation of autophagic vesicles and autophagosomes in mouse SPCs. Subsequently, a connection of autophagy and SPCs maintenance and survival was demonstrated in a dose-dependent manner. Moreover, mTOR was identified as an essential factor for autophagy in SPCs with a complicated mechanism: (1) mTOR is phosphorylated by AKT to activate its target genes, p70s6 kinase, resulting in the inhibition of autophagy during short-term busulfan treatment. (2) mTOR mediates autophagy with p53 together, to regulate the fate of SPCs. Collectively, observations from this study indicate that moderate autophagy effectively protects SPCs from the stress of chemotherapy, which may provide an important hint for fertility protection in clinic.
The Primary Scarring Alopecias are characterised by the irreversible destruction and fibrosis of hair follicles, leading to permanent and often disfiguring loss of hair. The pathophysiology of these diseases is not well understood. However, follicular-fibrosis and loss of the stem-cell niche appears to be a common theme. This review explores the pathogenesis of primary scarring alopecias, asking what happens to the stem cells of the hair follicle and how they may contribute to the progression of these diseases. Bulge-resident cells are lost (leading to loss of capacity for hair growth) from the follicle either by inflammatory-mediate apoptosis or through epigenetic reprogramming to assume a mesenchymal-like identity. What proportion of bulge cells is lost to which process is unknown and probably differs depending on the individual PCA and its specific inflammatory cell infiltrate. The formation of fibroblast-like cells from follicular stem cells may also mean that the cells of the bulge have a direct role in the pathogenesis. The identification of specific cells involved in the pathogenesis of these diseases could provide unique diagnostic and therapeutic opportunities to prevent disease progression by preventing EMT and specific pro-fibrotic signals.
Mesenquimal stem-cells (MSCs): functions and differentiation process. Transdifferentiation is the ability of MSCs to differentiate in various types of cells. Stem cells respond to stimulatory and inhibitory factors which they are subjected to, and by the inhibition and/or activation of certain molecular pathways, MSCs give rise to new tissue-specific cells. Homing happens when the MSCs moves to an injured tissue endothelium atracted by the chemoattractant effect from the citokynes released by the tissue injuries. The release of paracrine factors are the main mechanisms for the MSCs therapeutic action, since these factors contribute for pro-angiogenic, anti-fibrotic, anti-apoptotic, and immunomodulatory functions. Starting from the isolation of a tissue specific MSCs – from skin, lungs, liver, placenta or adipose tissue – from mouse or human, this cells can be expanded and enriched by various methods in vitro. bFGF promotes the expansion of MSCs. During the MSCs expansion, positive markers (CD105, CD73, CD90 and others) and negative markers (CD45, CD34, CD14 ou CD11b, CD79 ou CD19, HLA-DR and others) are used to classify this cell population. In addiction to self renewel in vitro, the MSCs can differentiate between osteoblasts, chondrocytes, adipocytes and other cell types. MSCs differentiation can be enhanced by specific-lineage factors such as Sox-9 VEGF, TGF-β1, insulin, dexamethasone, acid ascorbic, β-catenin and others factors
CRISPR immunity: acquisition, crDNA biogenesis and targeting. CRISPR loci’s clusters of repeats (black diamonds) and spacers (colored boxes) flanked by L (leader sequence) and CRISPR associated genes (cas). Following a unknown mechanism, in adaptation phase, the virus incorpores new spacers from his genome on the CRISPR array. The synthesis of a new repeat is also required. The crRNA transcribed from these CRISPR loci (biogenesis phase) associates with the Cas9 protein, a nuclease enzyme capable of cleaving DNA, and then the CrRNA-Cas9 associates to the trans-activating CRISPR RNA (tracrRNA), transcribed from an adjacent sequence to CRISPR. During the assembly of the CRISPR-Cas system, the tracrRNA and the crRNA combine to form a single strand called the guiding RNA (gRNA). Due to gRNA complementarity to the sequence containing the viral genome in the DNA, CRISPR-Cas binds accurately to the target (targeting phase)
Genetic engineering through the CRISPR-Cas system and Cas applications. (A) First of all, a guiding RNA (gRNA), complementary to the DNA sequence to be modified, must be designed. This gRNA will direct the gRNA-Cas complex to the DNA target region, complementary to its sequence of nitrogenous base pairs. The gRNA-Cas causes a double-strand DNA break. The genomic edition can occur through the non homologous end joining (NHEJ), producing variable length mutation (insertion or deletion); or through the homology directed repair (HDR), promoting precise length mutation. Cas applications: (B) Indel mutations. (C) Specific sequence insertion or replacement. (D) Large deletions or rearrangements. (E) Gene activation (reversible activation of coding/non-coding genes). (F) Histone modifications (transcription speed/rate of a given locus)
Mesenchymal stem cells (MSCs), also known as multipotent mesenchymal stromal stem cells, are found in the perivascular space of several tissues. These cells have been subject of intense research in the last decade due to their low teratogenicity, as well as their ability to differentiate into mature cells and to secrete immunomodulatory and trophic factors. However, they usually promote only a modest benefit when transplanted in experimental disease models, one of the limitations for their clinical application. The CRISPR-Cas system, in turn, is highlighted as a simple and effective tool for genetic engineering. This system was tested in clinical trials over a relatively short period of time after establishing its applicability to the edition of the mammalian cell genome. Similar to the research evolution in MSCs, the CRISPR-Cas system demonstrated inconsistencies that limited its clinical application. In this review, we outline the evolution of MSC research and its applicability, and the progress of the CRISPR-Cas system from its discovery to the most recent clinical trials. We also propose perspectives on how the CRISPR-Cas system may improve the therapeutic potential of MSCs, making it more beneficial and long lasting.
The lack of clear regulations for the use of veterinary stem cells has triggered the commercialization of unproven experimental therapies for companion animal diseases. Adult stem cells have complex biological characteristics that are directly related to the therapeutic application, but several questions remain to be answered. In order to regulate the use of these cells, well-conducted, controlled scientific studies that generate high-quality data should be performed, in order to assess the efficacy and safety of the intended treatment. This paper discusses the scientific challenges of mesenchymal stem cell therapy in veterinary regenerative medicine, and reviews published trials of adipose-tissue-derived stem cells in companion animal diseases that spontaneously occur.
Fetal-maternal microchimerism describes the acquisition of fetal stem cells (FSC) by the mother during pregnancy and their long-term persistence after parturition. FSC may engraft in a variety of maternal tissues especially if there is organ/tissue injury, but their role and mechanism of persistence still remains elusive. Clinical applications due to their pluripotency, immunomodulatory effects and accessibility make them good candidates for ex-vivo manipulation and autologous therapies. The hair follicles contain a distinctive niche for pluripotent stem cells (PSC). To date, there is no published evidence of fetal microchimerism in the hair follicle. In our study, follicular unit extraction (FUE) technique allowed easy stem cell cultures to be obtained while simple hair follicle removal by pull-out technique failed to generate stem cells in culture. We identified microchimeric fetal stem cells within the primitive population of maternal stem cells isolated from the hair follicles with typical mesenchymal phenotype, expression of PSC genes and differentiation potential towards osteocytes, adypocites and chondrocytes. This is the first study to isolate fetal microchimeric stem cells in adult human hair long after parturition. We presume a sanctuary partition mechanism with PSC of the mother deposited during early embryogenesis could explain their long-term persistence.
Illustration showing the percentage occupancy and functions displayed by N6-methyladenosine (m⁶A), N1 methyladenosine (m¹A), 5-methylcytosine (m⁵C) and pseudouridine (Ψ) modifications in eukaryotic mRNAs [20]
RNA base modification pathways: Adenine, cytosine and Uracil undergo base modifications, many of which are reversible in nature. Adenine can be modified by the enzymatic activity of methyltransferases (METTL3-METTL14) generating m⁶A, which can in turn be reversed by its specific erasers such as ALKBH5/FTO. m¹A can arise from adenine through damages in RNA, while the reversal is facilitated by ALKBH3. m¹A can also be dynamically converted to m⁶A by Dimroth rearrangement. Moreover, adenine also undergoes base editing by deaminases such as ADAR1 and ADAR2, converting it to hypoxanthine. Methyltransferases NSUN2 or TRDMT1 converts cytosine to m⁵C by methylating at position 5. m⁵C can further undergo enzymatic oxidation by TET dioxygeneses to generate hm⁵C, which in turn revert to cytosine with f⁵C (formyl group at cytosine 5). Cytosine can also be edited by a complex of proteins such as APOBEC1, RBM47 and A1CF into uracil. Finally, Uracil can undergo base isomerisation to pseudouracil by proteins such as PUS and H/ACA box ribonucleoproteins
Epitranscriptome code of a gene across cell types operational during determination of cell fate. RNA transcripts that are expressed in a cell type despite being same might be dynamically regulated by an epitranscriptome code mediated by different modification enzymes (writers, readers, and erasers). These further dictate stability, localisation as well as translation, enabling spatio-temporal expression of a gene in a cell type
m⁶A and its effectors regulates cell fate specification. During cellular differentiation, RNA m⁶A levels in cell type specific mRNAs are dynamically regulated by “writers”, “readers” and “erasers”. m⁶A methylation is considered as a ubiquitous modification in mRNAs, and is catalyzed by methyltransferases such as Mettl3/Mettl14; while FTO is responsible for the demethylation of m⁶A. There occurs a mutual interplay between RNA methylation/ demethylation and reader proteins such as Ythdc2, Ythdf2 functionally recognize and interpret m⁶A modifications during cell fate switching. This information is further essential for proper splicing, transportation, translation and stability of the mRNA transcripts, which forms the signature of a cell type. m⁷G: 7 Methylguanosine cap; AAAn: Poly-A tail
Schematic illustration depicting key effectors and relevance of RNA modification in regulating various developmental events
Precise regulation of transcriptome modulates several vital aspects in an organism that includes gene expression, cellular activities and development, and its perturbation ensuing pathological conditions. Around 150 post-transcriptional modifications of RNA have been identified till date, which are evolutionarily conserved and likewise prevalent across RNA classes including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and detected less frequently in small nuclear RNA (snRNA) and microRNAs (miRNA). Among the RNA modifications documented, N6-methyladenosine (m⁶A) is the best characterised till date. Also, N¹-methyladenosine (m¹A), 5-methylcytosine (m⁵C) and pseudouridine (Ψ) are some of the other prominent modifications detected in coding and non-coding RNAs. “Epitranscriptome”, ensemble of these post-transcriptional RNA modifications, precisely coordinates gene expression and biological processes. Current literatures suggest the critical involvement of epitranscriptomics in several organisms during early development, contributing to cell fate specification and physiology. Indeed, epitranscriptomics similar to DNA epigenetics involves combinatorial dynamics provided by modified RNA molecules and associated protein complexes, which function as “writers”, “erasers” and “readers” of these modifications. A novel code orchestrating gene expression during cell fate determination is generated by the coordinated effects of different classes of modified RNAs and its regulator proteins. In this review, we summarize the current knowhow on N6-methyladenosine (m⁶A), 5-methylcytosine (m⁵C) and pseudouridine (ψ) modifications in RNA, the associated regulator proteins and enumerate how the epitranscriptomic regulations are involved in cell fate determination.
While the fundamental mechanism by which cardiac cell therapy mitigates ventricular dysfunction in the post ischemic heart remains poorly defined, donor cell paracrine signaling is presumed to be a chief contributor to the afforded benefits. Of the many bioactive molecules secreted by transplanted cells, extracellular vesicles (EVs) and their proteinaceous, nucleic acid, and lipid rich contents, comprise a heterogeneous assortment of prospective cardiotrophic factors-whose involvement in the activation of endogenous cardiac repair mechanism(s), including reducing fibrosis and promoting angiogenesis, have yet to be fully explained. In the current study we aimed to interrogate potential mechanisms by which cardiac mesenchymal stromal cell (CMC)-derived EVs contribute to the CMC pro-angiogenic paracrine signaling capacity in vitro. Vesicular transmission and biological activity of human CMC-derived EVs was evaluated in in vitro assays for human umbilical vein endothelial cell (HUVEC) function, including EV uptake, cell survival, migration, tube formation, and intracellular pathway activation. HUVECs incubated with EVs exhibited augmented cell migration, tube formation, and survival under peroxide exposure; findings which paralleled enhanced activation of the archetypal pro-survival/pro-angiogenic pathways, STAT3 and PI3K-AKT. Cytokine array analyses revealed preferential enrichment of a subset of prototypical angiogenic factors, Ang-1 and Ang-2, in CMC EVs. Interestingly, pharmacologic inhibition of Tie2 in HUVECs, the cognate receptors of angiopoietins, efficiently attenuated CMC-EV-induced HUVEC migration. Further, in additional assays a Tie2 kinase inhibitor exhibited specificity to inhibit Ang-1-, but not Ang-2-, induced HUVEC migration. Overall, these findings suggest that the pro-angiogenic activities of CMC EVs are principally mediated by Ang-1-Tie2 signaling.
Aspirated follicular cells (AFCs) from the in vitro fertilization program can express various stem cell markers and are even able to differentiate into different types of cells in vitro. The female reproductive potential decreases with increasing age due to lowered ovarian reserve and oocyte quality, but data on the effect of female age on stem cell characteristics of AFCs are scarce. Therefore, the aim of this study was to elucidate whether female age affects the mesenchymal stem cell (MSC) characteristics of AFCs. Follicular aspirates were collected from 12 patients included in the in vitro fertilization programme with a normal ovarian reserve. Patients were divided into four age groups: Group A ≤ 30 years, Group B 31–35 years, Group C 36–39 years and Group D ≥ 40 years. After removal of the oocytes, AFCs were collected from follicular aspirates using hypo-osmotic technique and cultured in vitro, and their stemness was compared according to female age. The cultured AFCs were analysed for gene expression using the Human Mesenchymal Stem Cell RT² Profiler™ PCR Array, for their potential for differentiation into adipogenic and osteogenic lineage, and for their expression of MSC-related markers using immunocytochemistry. We found that female age can significantly influence their stemness: expression of pluripotency and MSC-related genes, and their differentiation potential. Despite the relatively high expression of MSC-related genes, the AFCs of the oldest patients had the lowest potential to differentiate into osteogenic and adipogenic lineages in vitro, which may be related to their age and the changed ovarian function.
Premature ovarian insufficiency (POI), a fertility disorder affecting women under 40 years of age, is characterized by early loss of ovarian function. This study was aimed to maintain ovarian function in POI animal models by mesenchymal stem cells (MSCs) transplantation with/without the supplementation of platelet-rich plasma (PRP). Adipose tissue-derived MSCs were isolated from inbred rats (Fisher-344), and constitutive expression of both VEGF and GFP were maintained by transfection with plasmids, pVEGF and pGFP-N. PRP was derived from the blood of healthy untreated rats. A total of 60 rats were divided into 5 groups of 12 rats in each. First group was kept as untreated-control (Control), and POI model was induced in Fisher-344 rats by cyclophosphamide in the next four groups. Second group was kept as sham-operated-control (Sham). MSC, PRP and MSC+ PRP-treated groups were transplanted following the validation of POI model in rats. After 2 months following the transplantation, anti-mullerian-hormone (AMH) and oestradiol (E2) blood levels were measured. Follicles were evaluated after hematoxylin-eosin staining, and the immunofluorescence staining and gene expression analyses were performed to show the ovarian regeneration. The follicular count was improved after MSC- and MSC + PRP-treatment to 63% of Control-group and significantly higher than that in Sham-group, but a significant increase was not observed in PRP-group. Higher AMH and E2 levels were measured in MSC + PRP than in Sham-group, and CXCL12, BMP-4, TGF-β and IGF-1 expressions were also increased. This study showed MSCs +/-PRP transplantation after POI supports recovery of the follicular count and function. For ovarian recovery, a single administration of PRP was found not sufficient. Although MSC treatment increased follicular regeneration, better results were obtained in the co-transplantation of MSCs and PRP. These results might be promising for follicular regeneration in POI patients.
The inflammasome as a gear or cogwheel that couples purinergic signaling with the complement cascade (ComC) in sterile inflammation of the brain. Increase of extracellular ATP in the brain tissue in response to stressors (1) activates via P2X7 receptor microglia that respond by activation of Nlrp3 inflammasome (2). As result of inflammasome activation several DAMPs are released, including Hmgb-1 and S100a9 (3), which are recognized by circulating mannan binding lectin (MBL) (4) and activate the ComC in the MBL-dependent pathway. Activation of the ComC leads to release of C5 cleavage fragments that are crucial to maintain inflammation state in the brain parenchyma (5)
The most important steps in the intracellular activation of NRLP3 inflammasome by extracellular ATP. Extracellular ATP activates P2X7 on microglia cells (step 1) which subsequently activates K+ efflux channel TWIK-2 (step 2). A decrease in K+ intracellular levels triggers the activation of the NRLP3 inflammasome complex (step 3). In response to this caspase, 1 cleaves pro-IL-1β and IL-18 to active ready for secretion IL-1β and IL-18 (step 4), and in addition cleaves gasdermin that releases N-gasdermin (Step 5) that insert into the cell membrane to create pores (step 6) for the release of IL-1β and IL-18 (step 7) as well as DAMPs (step 8)
The interplay between purinergic signaling and ComC activation during sterile brain inflammation. Increase in extracellular ATP secreted level in brain tissue stimulates via P2X7 receptor Nlrp3 inflammasome in microglia cells. Activated microglia secrete IL-1, IL-18 and ROS that promote sterile inflammation in brain parenchyma. Microglia cells also release HMGB1 and S100a9 that as DAMPs activate MBL pathway of ComC activation. Release of ComC proteins cleavage fragments such as C3a and C5a anaphylatoxins maintains sterile inflammation state of brain
Crosstalk between sterile inflammation of brain and bone marrow. Several pro-mobilizing factors released form brain during sterile inflammation stimulate bone marrow to release monocytes, HSPCs, MSCs, EPCs and VSELs. Some of these cells may enter brain parenchyma due to damaged and leaky brain-blood barrier
Recent evidence indicates that the occurrence of psychiatric disorders in patients is linked to a local “sterile” inflammation of brain or due to a systemic inflammation process that affects the central nervous system. This is supported by the observation that in peripheral blood of psychotic patients are detectable several mediators and markers of inflammation as well as clinical data on correlations between systemic chronic inflammatory processes and psychiatric disorders. This may explain why some reported anti-inflammatory treatment strategies have beneficial effects on ameliorating psychotic events. In this review we will present a concept that aberrant purinergic signaling and increases in extracellular level of adenosine triphosphate (ATP) in the brain parenchyma may lead to activation of Nlrp3 inflammasome in microglia cells and as a consequence microglia released danger associated molecular pattern (DAMP) proteins activate complement cascade (ComC) in mannan binding lectin (MBL) – dependent manner. Activation of ATP-Nlrp3 inflammasome-ComC axis may also orchestrate trafficking of stem cells released from bone marrow into peripheral blood observed in psychotic patients. Based on this, the ATP-Nlrp3 inflammasome-ComC axis may become a target for new therapeutic approaches, which justifies the development and clinical application of efficient anti-inflammatory treatment strategies targeting this axis in psychiatry.
The ionotropic P2X7 receptor (P2X7R) is involved in bone homeostasis but its role in osteogenesis is controversial. Thus, we investigated the expression of P2X7R and the effects exerted by its modulation in mesenchymal stromal cells from human subcutaneous adipose tissue (S-ASCs), which have potential therapeutic application in bone regenerative medicine. We found that undifferentiated S-ASCs expressed P2X7R and its functional splice variants P2X7AR and P2X7BR. Cell stimulation by P2X7R agonist BzATP (100 μM) neither modified proliferation nor caused membrane pore opening while increasing intracellular Ca²⁺ levels and migration. The P2X7R antagonist A438079 reversed these effects. However, 25-100 μM BzATP, administered to S-ASCs undergoing osteogenic differentiation, dose-dependently decreased extracellular matrix mineralization and expression of osteogenic transcription factors Runx2, alkaline phosphatase and osteopontin. These effects were not coupled to cell proliferation reduction or to cell death increase, but were associated to decrease in P2X7AR and P2X7BR expression. In contrast, expression of P2X7R, especially P2X7BR isoform, significantly increased during the osteogenic process. Noteworthy, the antagonist A438079, administered alone, at first restrained cell differentiation, enhancing it later. Accordingly, A438079 reversed BzATP effects only in the second phase of S-ASCs osteogenic differentiation. Apyrase, a diphosphohydrolase converting ATP/ADP into AMP, showed a similar behavior. Altogether, findings related to A438079 or apyrase effects suggest an earlier and prevailing pro-osteogenic activity by endogenous ATP and a later one by adenosine derived from endogenous ATP metabolism. Conversely, P2X7R pharmacological stimulation by BzATP, mimicking the effects of high ATP levels occurring during tissue injuries, depressed receptor expression/activity impairing MSC osteogenic differentiation.
Stem cells at the origin of endothelial progenitor cells and in particular endothelial colony forming cells (ECFCs) subtype have been largely supposed to be positive for the CD133 antigen, even though no clear correlation has been established between its expression and function in ECFCs. We postulated that CD133 in ECFCs might be expressed intracellularly, and could participate to vasculogenic properties. ECFCs extracted from cord blood were used either fresh (n = 4) or frozen (n = 4), at culture days <30, to investigate the intracellular presence of CD133 by flow cytometry and confocal analysis. Comparison with HUVEC and HAEC mature endothelial cells was carried out. Then, CD133 was silenced in ECFCs using specific siRNA (siCD133-ECFCs) or scramble siRNA (siCtrl-ECFCs). siCD133-ECFCs (n = 12), siCtrl-ECFCs (n = 12) or PBS (n = 12) were injected in a hind-limb ischemia nude mouse model and vascularization was quantified at day 14 with H&E staining and immunohistochemistry for CD31. Results of flow cytometry and confocal microscopy evidenced the positivity of CD133 in ECFCs after permeabilization compared with not permeabilized ECFCs (p < 0.001) and mature endothelial cells (p < 0.03). In the model of mouse hind-limb ischemia, silencing of CD133 in ECFCs significantly abolished post-ischemic revascularization induced by siCtrl-ECFCs; indeed, a significant reduction in cutaneous blood flows (p = 0.03), capillary density (CD31) (p = 0.01) and myofiber regeneration (p = 0.04) was observed. Also, a significant necrosis (p = 0.02) was observed in mice receiving siCD133-ECFCs compared to those treated with siCtrl-ECFCs. In conclusion, our work describes for the first time the intracellular expression of the stemness marker CD133 in ECFCs. This feature could resume the discrepancies found in the literature concerning CD133 positivity and ontogeny in endothelial progenitors.
Despite considerable advances made in understanding of lung cancer biology, there has been meek improvement in lung cancer treatment outcome with 4% to 5% increase in 5-year survival rates in the last four decades. Underlying problem of lung cancer recurrence and poor prognosis is attributed to the presence of cancer stem cells (CSCs) which possess the potential to differentiate, proliferate and trigger chemo-resistance, tumor progression and metastasis, despite initial elimination of the tumor. To address specific targeting of CSCs, we investigated the effects of a small molecule Verrucarin J (VJ) on lung cancer cell lines A549 and H1793. VJ significantly inhibited cell proliferation of both cell lines, with IC50 values of approximately 10 nM for A549 and 20 nM for H1793 respectively after 48 h of treatment. A549 cell line when treated with VJ, induced cell apoptosis with concomitant down regulation of key CSC specific genes- ALDH1, LGR5, OCT4 and CD133 in a dose-dependent manner. To delineate the molecular mechanism by which VJ targets lung cancer cells and CSCs, we determined the effects of VJ on CSC self-renewal pathways Wnt1/β-catenin and Notch1. Treatment of A549 cell line with VJ inhibited significantly both the signalling pathways, suggesting inhibition of expression of CSC genes by VJ through the inhibition of CSC self-renewal signalling pathways. Taken together, our results suggest that VJ may serve as a potent anticancer drug to target cancer cells and CSCs.
Mobilization of stem cells from bone marrow (BM) into peripheral blood (PB) in response to tissue or organ injury, infections, strenuous exercise, or mobilization-inducing drugs is as we postulated result of a “sterile inflammation” in the BM microenvironment that triggers activation of the Complement Cascade (ComC). Therefore, we became interested in the role of the Nlrp3 inflammasome in this process and show for the first time that its activation in ATP-dependent manner orchestrates BM egress of hematopoietic stem/progenitor cells (HSPCs) as well as other stem cells, including mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs). To explain this extracellular ATP is a potent activator of the Nrlp3 inflammasome, which leads to the release of interleukin 1β and interleukin 18, as well as several danger-associated molecular pattern molecules (DAMPs) that activate the mannan-binding lectin (MBL) pathway of the ComC, from cells of the innate immunity network. In support of this mechanism, we demonstrate that the Nlrp3 inflammasome become activated in innate immunity cells by granulocyte colony stimulating factor (G-CSF) and AMD3100 in an ATP-dependent manner. Moreover, administration of the Nlrp3 inflammasome activator nigericin induces mobilization in mice, and the opposite effect is obtained by administration of an Nlrp3 inhibitor (MCC950) to mice mobilized by G-CSF or AMD3100. In summary, our results further support the crucial role of innate immunity, BM sterile inflammation, and novel role of the ATP–Nlrp3–ComC axis in the egress of stem cells into PB.
Since last two decades, the major cancer research has focused on understanding the characteristic properties and mechanism of formation of Cancer stem cells (CSCs), due to their ability to initiate tumor growth, self-renewal property and multi-drug resistance. The discovery of the mechanism of acquisition of stem-like properties by carcinoma cells via epithelial-mesenchymal transition (EMT) has paved a way towards a deeper understanding of CSCs and presented a possible avenue for the development of therapeutic strategies. In spite of years of research, various challenges, such as identification of CSC subpopulation, lack of appropriate experimental models, targeting cancer cells and CSCs specifically without harming normal cells, are being faced while dealing with CSCs. Here, we discuss the biology and characteristics of CSCs, mode of acquisition of stemness (via EMT) and development of multi-drug resistance, the role of tumor niche, the process of dissemination and metastasis, therapeutic implications of CSCs and necessity of targeting them. We emphasise various strategies being developed to specifically target CSCs, including those targeting biomarkers, key pathways and microenvironment. Finally, we focus on the challenges that need to be subdued and propose the aspects that need to be addressed in future studies in order to broaden the understanding of CSCs and develop novel strategies to eradicate them in clinical applications. Graphical AbstractCancer Stem Cells(CSCs) have gained much attention in the last few decades due to their ability to initiate tumor growth and, self-renewal property and multi-drug resistance. Here, we represent the CSC model of cancer, Characteristics of CSCs, acquisition of stemness and metastatic dissemination of cancer, Therapeutic implications of CSCs and Various strategies being employed to target and eradicate CSCs.
Beneficial effects of MSC-based treatments for various chemotherapy-induced tissue injuries. Biological effects of MSC administration observed in preclinical and clinical trials on various tissue damages caused by chemotherapy.
MSCs‘ tissue of origin in preclinical models for chemotherapy-induced side effects. Diagrams show the percentage of the MSCs’ tissue of origin in preclinical models for chemotherapy-induced tissue toxicities.
Chemotherapy constitutes one of the key treatment modalities for solid and hematological malignancies. Albeit being an effective treatment, chemotherapy application is often limited by its damage to healthy tissues, and curative treatment options for chemotherapy-related side effects are largely missing. As mesenchymal stromal cells (MSCs) are known to exhibit regenerative capacity mainly by supporting a beneficial microenvironment for tissue repair, MSC-based therapies may attenuate chemotherapy-induced tissue injuries. An increasing number of animal studies shows favorable effects ofMSC-based treatments; however, clinical trials for MSC therapies in the context of chemotherapy-related side effects are rare. In this concise review, we summarize the current knowledge of the effects of MSCs on chemotherapy-induced tissue toxicities. Both preclinical and early clinical trials investigating MSC-based treatments for chemotherapy-related side reactions are presented, and mechanistic explanations about the regenerative effects of MSCs in the context of chemotherapy-induced tissue damage are discussed. Furthermore, challenges of MSC-based treatments are outlined that need closer investigations before these multipotent cells can be safely applied to cancer patients. As any pro-tumorigenicity of MSCs needs to be ruled out prior to clinical utilization of these cells for cancer patients, the pro- and anti-tumorigenic activities of MSCs are discussed in detail.
Retinal diseases were always difficult problem for clinical ophthalmology. Modern methods of their treatment only decrease risk of complications, however in Russia was created better technology for this purpose: peptide bioregulators, which were made by sequential adding of amino acids one to another, binding with the promoter region of genes, and promoting retinoprotective effect by regulation of their expression, improving the state of the retina. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.
Ossification process of the long bone in mammals [19]
Characterization of CSPCs isolated from human, equine and bovine articular cartilage. a expression of stem-cell-related surface markers; b Properties of CSPCs and use for osteoarthritis (OA) management [41]
A schematic representation of the development of the osteochondrosis lesions complex in horses and the associated factors involved in pathogenesis [1]
Osteochondrosis (osteochondrosis dissecans; OCD) is a disease syndrome of growing cartilage related to different clinical entities such as epiphysitis, subchondral cysts and angular carpal deformities, which occurs in growing animals of all species, including horses. Nowadays, these disorders are affecting increasing numbers of young horses worldwide. As a complex multifactorial disease, OCD is initiated when failure in cartilage canals because of existing ischemia, chondrocyte biogenesis impairment as well as biochemical and genetic disruptions occur. Recently, particular attention have been accorded to the definition of possible relations between OCD and some metabolic disorders; in this way, implication of mitochondrial dysfunctions, endoplasmic reticulum disruptions, oxidative stress or endocrinological affections are among the most considered axes for future researches. As one of the most frequent cause of impaired orthopaedic potential, which may result in a sharp decrease in athletic performances of the affected animals, and lead to the occurrence of complications such as joint fragility and laminitis, OCD remains as one of the primary causes of considerable economic losses in all sections of the equine industry. It would therefore be important to provide more information on the exact pathophysiological mechanism(s) underlying early OC(D) lesions, in order to implement innovative strategies involving the use of progenitor stem cells, which are considered nowadays as a promising approach to regenerative medicine, with the potential to treat numerous orthopaedic disorders, including osteo-degenerative diseases, for prevention and reduction of incidence of the disease, not only in horses, but also in human medicine, as the equine model is already widely accepted by the scientific community and approved by the FDA, for the research and application of cellular therapies in the treatment of human conditions.
Gestational diabetes mellitus (GDM) has been associated with an increased risk of maternal and neonatal morbidity. The Wharton’s jelly (WJ) of the umbilical cord (UC) is a useful indicator of the deleterious effects of hyperglycemia on fetal tissues as it represents the fetus embryologically, physiologically and genetically. We studied WJ mesenchymal stem cells (hWJSCs) from UC from mothers without GDM (Normal; n = 3); insulin-controlled GDM mothers (GDMi; n = 3) and diet-controlled GDM mothers (GDMd; n = 3)]. Cell proliferation, stemness markers, telomerase, osteogenic and chondrogenic differentiation, antioxidant enzymes and gene expression for mitochondrial function (ND2, TFAM, PGC1α, and NDUFB9) were significantly lower in GDMi-hWJSCs and GDMd-hWJSCs compared to normal hWJSCs (P < 0.05). On the other hand, cell cycle inhibitors (p16, p21, p27) and p53 were remarkably up-regulated in GDMi-hWJSCs and GDMd-hWJSCs compared to normal hWJSCs. The results from this study confirmed that maternal hyperglycemia even though managed with insulin or diet, induced changes in the properties of the WJ and its cells. These changes may also be observed in fetal tissues and if true, prevention of the onset of gestational diabetes should be a priority over management. Generation of tissues that simulate those of the fetus such as pancreatic and cardiovascular cells from GDM-hWJSCs by direct differentiation or via induced pluripotent stem cell reprogramming provide possible platforms to evaluate the effects of glucose on specific fetal organ.
Background Acute myocardial infarction (AMI) and the ensuing ischemic heart disease are approaching an epidemic state. Limited stem cell retention following intracoronary administration has reduced the clinical efficacy of this novel therapy. Polymer based cell coating is biocompatible and has been shown to be safe. Here, we assessed the therapeutic utility of gelatin-based biodegradable cell coatings on bone marrow derived cell retention in ischemic heart. Methods Gelatin based cell coatings were formed from the surface-mediated photopolymerization of 3% gelatin methacrylamide and 1% PEG diacrylate. Cell coating was confirmed using a multimodality approach including flow cytometry, imaging flow cytometry (ImageStream System) and immunohistochemistry. Biocompatibility of cell coating, metabolic activity of coated cells, and the effect of cell coating on the susceptibility of cells for engulfment were assessed using in vitro models. Following myocardial infarction and GFP+ BM-derived mesenchymal stem cell transplantation, flow cytometric and immunohistochemical assessment of retained cells was performed. Results Coated cells are viable and metabolically active with coating degrading within 72 h in vitro. Importantly, cell coating does not predispose bone marrow cells to aggregation or increase their susceptibility to phagocytosis. In vitro and in vivo studies demonstrated no evidence of heightened immune response or increased phagocytosis of coated cells. Cell transplantation studies following myocardial infarction proved the improved retention of coated bone marrow cells compared to uncoated cells. Conclusion Gelation based polymer cell coating is biologically safe and biodegradable. Therapies employing these strategies may represent an attractive target for improving outcomes of cardiac regenerative therapies in human studies.
Mesenchymal stem cells (MSCs) are currently being tested in several clinical trials. Mitochondria regulate many aspects of MSC function. Mitochondrial preproteins are rapidly translated and trafficked into the mitochondrion for assembly in their final destination, but whether coexisting cardiovascular risk factors modulate this process is unknown. We hypothesized that metabolic syndrome (MetS) modulates mitochondrial protein import in porcine MSCs. MSCs were isolated from porcine abdominal adipose tissue after 16 weeks of Lean or MetS diet (n = 5 each). RNA-sequencing was performed and differentially expressed mitochondrial mRNAs and microRNAs were identified and validated. Protein expression of transporters of mitochondrial proteins (presequences and precursors) and their respective substrates were measured. Mitochondrial homeostasis was assessed by Western blot and function by cytochrome-c oxidase-IV activity. Forty-five mitochondrial mRNAs were upregulated and 25 downregulated in MetS-MSCs compared to Lean-MSCs. mRNAs upregulated in MetS-MSCs encoded for precursor proteins, whereas those downregulated encoded for presequences. Micro-RNAs upregulated in MetS-MSCs primarily target mRNAs encoding for presequences. Transporters of precursor proteins and their substrates were also upregulated, associated with changes in mitochondrial homeostasis and dysfunction. MetS interferes with mitochondrial protein import, favoring upregulation of precursor proteins, which might be linked to post-transcriptional regulation of presequences. This in turn alters mitochondrial homeostasis and impairs energy production. Our observations highlight the importance of mitochondria in MSC function and provide a molecular framework for optimization of cell-based strategies as we move towards their clinical application.
There is a growing interest in the potential of adult stem cells for implementing regenerative medicine in the brain. We assessed the effect of intracerebroventricular (icv) administration of human umbilical cord perivascular cells (HUCPVCs) on spatial memory of senile (27 mo) female rats, using intact senile counterparts as controls. Approximately one third of the animals were injected in the lateral ventricles with a suspension containing 4.8 X 10⁵ HUCPVC in 8 μl per side. The other third received 4.8 X 10⁵ transgenic HUCPVC overexpressing Insulin-like growth factor-1 (IGF-1) and the last third of the rats received no treatment. Spatial memory performance was evaluated using a modified version of the Barnes maze test. In order to evaluate learning ability as well as spatial memory retention, we assessed the time spent (permanence) by animals in goal sector 1 (GS1) and 3 (GS3) when the escape box was removed. Fluorescence microscopy revealed the prescence of Dil-labeled HUCPVC in coronal sections of treated brains. The HUCPVC were located in close contact with the ependymal cells with only a few labeled cells migrating into the brain parenchyma. After treatment with naïve or IGF-1 transgenic HUCPVC, permanence in GS1 and GS3 increased significantly whereas there were no changes in the intact animals. We conclude that HUCPVC injected icv are effective to improve some components of spatial memory in senile rats. The ready accessibility of HUCPVC constitutes a significant incentive to continue the exploration of their therapeutic potential on neurodegenerative diseases.
Vascularization is a major hurdle in complex tissue and organ engineering. Tissues greater than 200 μm in diameter cannot rely on simple diffusion to obtain nutrients and remove waste. Therefore, an integrated vascular network is required for clinical translation of engineered tissues. Microvessels have been described as <150 μm in diameter, but clinically they are defined as <1 mm. With new advances in super microsurgery, vessels less than 1 mm can be anastomosed to the recipient circulation. However, this technical advancement still relies on the creation of a stable engineered microcirculation that is amenable to surgical manipulation and is readily perfusable. Microvascular engineering lays on the crossroads of microfabrication, microfluidics, and tissue engineering strategies that utilize various cellular constituents. Early research focused on vascularization by co-culture and cellular interactions, with the addition of angiogenic growth factors to promote vascular growth. Since then, multiple strategies have been utilized taking advantage of innovations in additive manufacturing, biomaterials, and cell biology. However, the anatomy and dynamics of native blood vessels has not been consistently replicated. Inconsistent results can be partially attributed to cell sourcing which remains an enigma for microvascular engineering. Variations of endothelial cells, endothelial progenitor cells, and stem cells have all been used for microvascular network fabrication along with various mural cells. As each source offers advantages and disadvantages, there continues to be a lack of consensus. Furthermore, discord may be attributed to incomplete understanding about cell isolation and characterization without considering the microvascular architecture of the desired tissue/organ.
Stroke remains a major unmet clinical need that warrants novel therapies. Following an ischemic insult, the cerebral vasculature secretes inflammatory molecules, creating the stroke vasculome profile. The present study evaluated the therapeutic effects of endothelial cells on the inflammation-associated stroke vasculome. qRT-PCR analysis revealed that specific inflammation-related vasculome genes BRM, IκB, Foxf1, and ITIH-5 significantly upregulated by oxygen glucose deprivation (OGD. Interestingly, co-culture of human endothelial cells (HEN6) with human endothelial cells (EPCs) during OGD significantly blocked the elevations of BRM, IκB, and Foxf1, but not ITIH-5. Next, employing the knockdown/antisense technology, silencing the inflammation-associated stroke vasculome gene, IκB, as opposed to scrambled knockdown, blocked the EPC-mediated protection of HEN6 against OGD. In vivo, stroke animals transplanted with intracerebral human EPCs (300,000 cells) into the striatum and cortex 4 h post ischemic stroke displayed significant behavioral recovery up to 30 days post-transplantation compared to vehicle-treated stroke animals. At 7 days post-transplantation, quantification of the fluorescent staining intensity in the cortex and striatum revealed significant upregulation of the endothelial marker RECA1 and a downregulation of the stroke-associated vasculome BRM, IKB, Foxf1, ITIH-5 and PMCA2 in the ipsilateral side of cortex and striatum of EPC-transplanted stroke animals relative to vehicle-treated stroke animals. Altogether, these results demonstrate that EPCs exert therapeutic effects in experimental stroke possibly by modulating the inflammation-plagued vasculome.
Transcriptional silencing ofXist. Protein LBR allows Xist to localize and spread throughout X chromosome by binding to RepA domain. Deacetylation of X chromosome is achieved by SHARP-SMRT-HDAC3 complex. To date, there is no direct evidence proving that SHARP is responsible for PRC1 and PRC2 association (represented by dash arrows). M, H3K37me3; ub, H2AK119ub; ac, histone acetylation
Dynamic process of dosage compensation in preimplantation embryos of mouse and human. In mouse preimplantation embryo (marked in green), dosage compensation is initiated in the four-cell stage by selectively inhibiting paternal X chromosome, wherein zygotic genome activation occurs. Tsix antagonizes Xist on activated X chromosome. During late blastocyst stage, epiblast cells are reactivated by upregulation of Tsix, while trophectoderm cells remain imprinted. After implantation, one of the two X chromosomes is inactivated by upregulation of Xist in epiblast cells. In human preimplantation embryo (marked in orange), imprinted X chromosome inactivation is lost. X-chromosome dampening (XCD), which induces biallelic transcriptional silencing, begins after zygotic genome activation during at eight-cell stage. This process is established by accumulation of Xist and Xact on both X chromosomes. During late blastocyst stage, expression of Xact declines. Xist is upregulated on one of the X chromosomes. Xp, paternal X chromosome; Xm, maternal X chromosome; TE, trophectoderm; PE, primitive endoderm
Various states of X chromosome in human embryonic stem cells (hESCs) and proposed model of cancer stem cells (CSCs). (a) Primed hESC differs from pre-implantation blastocyst based on mono-allelic expression of Xist on Xi. During in vitro culturing, XCE may occur and result in loss of Xist on Xi. In contrast, naïve hESCs resemble preimplantation blastocyst as both X chromosomes are activated. However, the predominant cell population shows mono-allelic expression of Xist (*, indicating major cell type), yet a small subset of naïve hESCs is completely identical to blastocysts with biallelic expression of Xist. X-chromosome status persists after passing to descendant somatic cells even without proper dosage compensation. In addition, an intermediate XaXa state exists transiently during the transition between primed hESC and naïve hESC. (b) Pluripotency factor (indicated by lightning symbol) reverses normal XaXi state in somatic cell to either primed state or naïve state cancer stem cell (CSC). During differentiation of primed state CSC, XCI occurs and downregulation of Xist leads to partial reactivation of X-linked oncogenes. In contrast, expression of Xist increases during primed-to-naïve transition, which silences X-linked tumor suppressor genes. It is important to note that improper dosage compensation will pass to daughter cells and promote tumor growth. (Note – the proposed model is based on the currently known stem cell theory. We hypothesize pluripotency factors induces transformation of cancer stem cell either by upregulating or downregulating Xist)
Long non-coding RNA (lncRNA) Xist has emerged as a key modulator in dosage compensation by randomly inactivating one of the X chromosomes in mammals during embryonic development. Dysregulation of X chromosome inactivation (XCI) due to deletion of Xist has been proven to induce hematologic cancer in mice. However, this phenomenon is not consistent in humans as growing evidence suggests Xist can suppress or promote cancer growth in different organs of the human body. In this review, we discuss recent advances of XCI in human embryonic stem cells and provide an explanation for the seemingly contradictory roles of Xist in development of human cancer.
Multipotent mesenchymal stem/stromal cells (MSCs) have regenerative and immunomodulatory properties to restore and repair injured tissues, making them attractive candidates for cell-based therapies. Experimental and clinical evidence has demonstrated the effectiveness of MSC transplantation in managing diabetes mellitus (DM). Autologous MSCs are assumed to be favorable because patient-derived cells are readily available and do not entail sustained immunosuppressive therapy. DM is associated with hyperglycemia, oxidative stress and altered immune responses and inflammation. It may thus alter the biological characteristics and therapeutic qualities of human MSCs (hMSCs). Several studies have explored the effect of DM or the diabetic microenvironment on the engraftment and efficacy of transplanted MSCs, which are determined by proliferation, differentiation, senescence, angiogenesis supportive effect, migration, anti-oxidative capacity and immunomodulatory properties. This review aims to present the available data on how DM impacts MSC biology and functionality and identify future perspectives for autologous MSC-based therapy in diabetics.
of the plot cellular and gene therapy in the regenerative medicine
Chart of clinical trials of cell and gene therapy. A search of the database of clinical trials ( for registered studies containing the term “Gene therapy” and “Cell therapy”. Pie chare of trials by clinical phase and Pie chart of the status of studies have been shown
The pace of advances in the world of science have created new opportunities and insights that give us new and more understanding of our nature and environment. Among the different fields of science, new medical sciences have drawn a great deal of attention among medical science researchers and the society. The hope for finding treatments for incurable diseases and further improvement of man’s health is growing thanks to new medical technologies. Among the novel medical fields that have been extensively covered by medical and academic societies are cell therapy and gene therapy that are categorized under regenerative medicine. The present paper is an attempt to introduce the prospect of a curative cell-based therapy and new cellular and gene therapy drugs that have been recently approved by FDA (food and drug administration). Cellular and gene therapy are two very close fields of regenerative medicine and sciences which their targets and applications can be discussed together. What adds to the importance of this new field of science is the possibility to translate the hope for treatment of incurable diseases into actual treatments. What follows delves deeper into this new field of science and the drugs.
Direct effects of CTX injection and MenSCs transplantation on ovarian changes and estrous cycles. a The macroscopical changes of ovaries in No, Mo and Me groups from top to bottom after ten days since MenSCs graft. b Changes of ovarian weight among the three groups, n = 20 (data were represented as mean ± SEM, **P < 0.01). c Changes of uterine weight among the three groups, n = 20 (data were represented as mean ± SEM, #P = 0.051, **P < 0.01). c Representative images of ovarian histological sections in No group (a), Mo group (b) and Me group (c). Original magnification, ×100. d The statistics of total follicles in each group, n = 6 (data were represented as mean ± SEM, **P < 0.01). e Changes in numbers of primordial, primary, secondary, antral and preovulatory follicles in each group, n = 6 (data were represented as mean ± SEM, #P = 0.056, *P < 0.05, **P < 0.01). f Representative images of estrous cycles in female mice including proestrus (d), estrus (e), metaestrus (f) and diestrus (g). Original magnification, ×100. g Variation trends of estrous cycles recorded every ten days until the fortieth day since MenSCs graft in No, Mo and Me groups, respectively, n = 6
Physiological changes to evaluate ovarian status. a Serum hormone levels assessed by ELISA kits in each group for measuring FSH (left), E2 (middle) and AMH (right), n = 6 (data were represented as mean ± SEM, *P < 0.05, **P < 0.01). b Representative images of parturition in each group after mating with male mice. c Records of live births after CTX injection followed by MenSCs transplantation, n = 6 (data were represented as mean ± SEM, **P < 0.01). d Apoptosis evaluation using Hoechst kit. Yellow arrows pointed to apoptotic cells with fragmented or condensed nuclei of apoptotic cells. Original magnification, ×100. e Apoptosis evaluation performed by TUNEL assay. White arrows pointed to FITC-labeled apoptotic cells. Original magnification, ×100. f Relative mRNA expression by q-RT PCR analysis for AMH, DDX4 and VEGFA controlled to GAPDH with fold change measured by 2-ΔΔCT, n = 6. g Relative expression at protein levels of AMH, DDX4 and VEGFA (upper panel) and quantitative analysis (lower panel)
DEGs detection with mRNA sequencing analysis. a Boxplot of RPKM value of each sample in Mo and Me groups, showing similar expression levels of genes. b Statistics of DEGs basing on P < 0.05 and fold change >2 or < 0.5 when comparing Mo group with Me group. c Statistical analysis result plot for DEGs as volcano plot. d Statistical analysis result plot for DEGs as M-A plot. e Hierarchical clustering group heatmap of DEGs screened on the basis of P < 0.05 and fold change >2 or < 0.5. Red revealed genes that were relatively up-regulated, while green indicated down-regulated genes and black meant not statistically significant
Go enrichment and signaling regulatory modules analysis of DEGs. a Go enrichment analysis including biological process, cellular component and molecular function. b KEGG pathway analysis of DEGs between Mo group and Me group. c Gene-co-expression network to analyze interaction of DEGs with r > 0.95 or < −0.95. The core genes were selected from DEGs which were involved in such pathways we concerned as ECM-receptor, focal adhesion and PI3K-AKT signaling pathways
Signaling pathway validation. a Relative mRNA expression by q-RT PCR analysis of extracellular molecules of COL6A5 and COL9A2 in No, Mo and Me groups, n = 6 (data were represented as mean ± SEM, #P = 0.052, **P < 0.01). b Western blotting analysis for COL6A5 and COL9A2 expression (upper panel) and quantitative analysis (lower panel). c IHC analysis for detecting the expression of COL6A5 and COL9A2. d Western blotting analysis for FAK, AKT, CDKN1A, NR4A1 and their phosphorylated proteins (left panel) and quantitative gray level analysis (right panel)
POI is characterized by “absent not abnormal” menstruation with hormonal disorders in woman younger than 40 years of age, and etiological and pathophysiological mechanisms underlying the POI development have not been clearly defined. Recently, due to advantages such as abundant sources and non-invasive methods of harvest, MenSCs have been emerging as a promising treatment strategy for the recovery of female reproductive damage. Here, we demonstrated that MenSCs graft in POI mice after CTX treatment could restore ovarian function by regulating normal follicle development and estrous cycle, reducing apoptosis in ovaries to maintain homeostasis of microenvironment and modulating serum sex hormones to a relatively normal status. Moreover, MenSCs participated in the activation of ovarian transcriptional expression in ECM-dependent FAK/AKT signaling pathway and thus restored ovarian function to a certain extent. MenSCs transplantation was proved to be an effective way to repair ovarian function with low immunogenicity, suggesting its great potential for POI treatment. Electronic supplementary material The online version of this article (10.1007/s12015-018-9867-0) contains supplementary material, which is available to authorized users.
Cell therapy in stroke: main sources, cell types, route of delivery, mechanisms identified so far in rodent (top) and evaluated in human (bottom). ESC, Embryonic Stem Cell; iPS, induced Pluripotent stem cell; VSEL, Very Small Embryonic-Like stem cells; MAPC, Multipotent Adult Progenitor Cells (purple, pluripotent cells). HSC, Hematopoietic Stem Cell; NPC, Neural Progenitor Cell; EPC, Endothelial Progenitor Cell; SMPC, Smooth Muscle Progenitor Cell; MSC, Mesenchymal Stem Cell (pink, multipotent cells) and MNC, Mononuclear Cell (orange, differentiated cells). IV, intravenous; IC, intracerebral; IA, intraarterial pathways. IL10, Interleukin-10; INFγ, Interferon gamma
Stroke is a major public health issue with limited treatment. The pharmacologically or mechanically removing of the clot is accessible to less than 10% of the patients. Stem cell therapy is a promising alternative strategy since it increases the therapeutic time window but many issues remain unsolved. To avoid a new dramatic failure when translating experimental data on the bedside, this review aims to highlight the indispensable checkpoints to make a successful clinical trial based on the current preclinical literature. The large panel of progenitors/ stem cells at the researcher’s disposal is to be used wisely, regarding the type of cells, the source of cells, the route of delivery, the time window, since it will directly affect the outcome. Mechanisms are still incompletely understood, although recent studies have focused on the inflammation modulation of most cells types.
MeCP2 expression after reprogramming
Immunocytochemistry for MeCP2 of a patient R255X line after viral reprogramming at ViPSC passage 1 (P1) and b passage 3 (P3); c patient RTT-FB R255X d patient RTT-FB R270X lines and e patient RTT-FB DEL at fibroblast stage and after episomal reprogramming (the EiPSC stage), respectively, at passage 12 and 11 (P12 + P11)
Generation of RTT and isogenic control cell line
Immunocytochemistry for MeCP2 of (a) manually-sorted RTT patient fibroblasts. Into MeCP2-positive (upper panels) and –negative (lower panels) cells, and (b) the EiPSC-derived lines. c PCR analysis of MeCP2 expression of cell lines EiPSC DEL CTR and EiPSC DEL MUT
H3K27me3 condensation andXISTexpression in early ViPSC and EiPSC passage lines
Immunocytochemistry for H3K27me3 of control iPSC lines at passages P4, P7, P10, P15 generated via a episomal reprogramming and b viral reprogramming. c RNA-Seq analysis of XIST for episomal and viral reprogrammed lines at passage P5 and P10 (two-sided t-test, n = 2, p = 0,04, data is represented at mean). PluriTest scores for all EiPSCs and ViPSCs
Embryoid Body formation
a Brightfield images of day 1 and 3 of embryoid bodies formed from iPSCs. b Immunocytochemistry of both ViPSC- derived EBs (upper panels) and EiPSC- derived EBs (lower panels) for all three germ layers: (i) α-Fetoprotein (endoderm), (ii) α-SMA (mesoderm) and (iii) β-III Tubulin (ectoderm)
Generation of proper controls is crucial in induced pluripotent stem cell (iPSC) studies. X-chromosomal disorders offer the potential to develop isogenic controls due to random X-chromosomal inactivation (XCI). However, the generation of such lines is currently hampered by skewed X-inactivation in fibroblast lines and X-chromosomal reactivation (XCR) after reprogramming. Here we describe a method to generate a pure iPSC population with respect to the specific inactivated X-chromosome (Xi). We used fibroblasts from Rett patients, who all have a causal mutation in the X-linked MeCP2 gene. Pre-sorting these fibroblasts followed by episomal reprogramming, allowed us to overcome skewness in fibroblast lines and to retain the X-chromosomal state, which was unpredictable with lentiviral reprogramming. This means that fibroblast pre-sorting followed by episomal reprogramming can be used to reliably generate iPSC lines with specified X-chromosomal phenotype such as Rett syndrome. Electronic supplementary material The online version of this article (10.1007/s12015-018-9851-8) contains supplementary material, which is available to authorized users.
Role of EPCs in neovasculogenesis: Tissue hypoxia leads to EPC mobilization from bone marrow to peripheral blood. Upon chemoattractant stimulation these cells are attracted to the zone of ischemia in a process called homing, they invade perivascular tissue and differentiate into mature endothelial cells or smooth muscle cells. TGF – tumour growth factor, smad-2 – signal transcriptional modulator, PDGF-BB – platelet-derived growth factor, eNOS – endothelial nitric oxide synthase, G-CSF – granulocyte colony-stimulating factor, SDF-1 – stromal cell-derived factor-1, VEGF – vascular endothelial growth factor, EPO – erythropoietin, IL-8 – interleukin 8, EPC – endothelial progenitor cell, EC – endothelial cell, SMA – smooth muscle cells
Endothelial progenitors are a population of cells with the inherent capacity to differentiate into mature endothelial cells and proangiogenic paracrine action. These characteristics have led to extensive studies being performed and tested in the treatment of tissue ischemia. The natural course of diabetes mellitus (DM) results in multiple areas of vascular damage. Thus endothelial progenitor cells‘(EPCs) beneficial potential is particularly desirable in diabetic patients. In this review, we summarize contemporary knowledge of EPC biology in DM. It has been shown that EPC functions are considerably impaired by DM. The presence of peripheral arterial disease (PAD) seems to further exacerbate the deficiencies of EPCs. However, studies examining EPC counts in PAD and DM observed disparate results, which can be due to a lack of consensus on precise EPC immunotype used in the different studies. Nevertheless, the results of EPC-based autologous cell therapy (ACT) are promising. In addition, EPCs have been shown to bean independent predictor of cardiovascular risk and diabetic foot ulcer healing.
Analysis of PD-L1+ lung cancer stem cells (CSCs)in lymph nodes aspirates by flow cytometry, a/ CSCs among other cells- marked in red, b/CSCs in red vs. lymphocytes in green, c/confirmation of non-lymphoid origin of CSCs by CD45 marker- CSCsin blue, lymphocytes in green, d/CD133 + EpCAM+cells gate, e/CSCs PD-L1 +
Comparison of the proportion of CD133 + EpCAM+PD-L1+ cells in metastatic LNs with free LNs. Difference significant in Mann Whitney test
Proportion of CD133 + EpCAM+PD-L1+ in LNs of patients with different histological types of lung cancer. SQCLC- squamous cell carcinoma, ADC- adenocarcinoma, NOS- not otherwise specified subtype
Proportion of CD133 + EpCAM+PD-L1+ in LNs of patients with IV stage of lung cancer vs. lower stages, difference not significant
Significant correlation between proportion of CD133 + EpCAM+PD-L1+ cells in LNs and pack years smoked (r = 0.4, p < 0.05)
Objectives An immunotherapy was found to be effective in achieving long-term survival in some lung cancer patients. It has emerged to searching for new immune biomarkers for select the best candidates to this therapy. It is suggested that cancer stem cells (CSCs) are responsible for tumor initiation, maintenance and its metastatic potential. However, a role of CSCs in escape of cancer from immunosurveillance is unknown. The aim of the study was assess the phenotype of putative CSCs and to examine the expression of PD-L1 on CSCs in metastatic lymph nodes (LNs) in lung cancer patients. Material and Methods Flow cytometry was used for CSCs evaluation in peripheral blood and EBUS/TBNA aspirates from N1,N2 lymph nodes in lung cancer patients. Results Of 30 patients the LNs metastases were confirmed in 18 patients. We noticed presence of PD-L1 on putative lung CSCs- CD133 + EpCAM+ cells. A higher percentage of CD133 + EpCAM+PD-L1+ cells was observed in patients with metastatic in LNs- median value = 4.38% than in patients without LNs metastases– median value = 0,015% (p < 0.05). The highest proportion of PD-L1+ CSCs was found in adenocarcinoma patients and in those with oncogene addiction what indicate an particular biology of this type of lung cancer. Conclusion The presence of CSCs with expression of PD-L1 in the metastatic LNs might suggest their immunogenic potential. EBUS/TBNA is commonly used in diagnosis and staging of lung cancer, so the analysis of the cells in metastatic LNs may fit in “immunoscoring” before immunotherapy.
T cell malignancies are aggressive diseases with no standard treatment available, often resulting in poor patient outcomes. Lately, the recent FDA approval of a CD19 CAR T cell therapy for B cell acute lymphoblastic leukemia has earned nationwide attention, leading to the possibility that success of CD19 CAR therapy can be extended to T cell malignancies. However, the impact of T cell depletion due to a shared antigen pool remains an issue to be resolved. Here, we describe a CD4CAR capable of eliminating CD4-positive T cell acute lymphoblastic leukemia in a systemic mouse model, with CAMPATH (alemtuzumab) as a natural safety switch to deplete the infused CD4CAR T cells to prevent toxicities associated with CD4 cell aplasia. Our data support the potential use of CD4CAR T cells for the treatment of CD4-postive T-cell acute lymphoblastic leukemia malignancies or refractory disease in clinical settings. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.
Stem cell aging underlies aging-associated disorders, such as steeply increased incidences of tumors and impaired regeneration capacity upon stress. However, whether and how the intestinal stem cells age remains largely unknown. Here we show that intestinal stem cells derived from 24-month-old mice hardly form typical organoids with crypt-villus structures, but rather mainly form big, rounded cysts devoid of differentiated cell types, which mimics the culturing of heterozygous APC-deficient cells from the APCmin mouse line. Further analysis showed that cultured crypts derived from aged mice exhibited reduced expression levels of differentiation genes and higher expression of Wnt target genes. Lowering the concentration of R-spondin-1 in the culture system significantly reduced formation of rounded cysts, accompanied by an increased formation of organoids from crypts derived from old mice. We are the first to uncover that intestinal stem cells derived from old mice harbor significant deficiency in differentiation that can be partially rescued through a reduction in R-spondin-1 exposure. This could be highly relevant to intestinal tumor development and the reduced regeneration potential observed in the aged population. Our study provides the first experimental evidence that an over-responsiveness to Wnt/beta-catenin signaling of aged intestinal stem cells mediates the aging-induced deficiency in differentiation, and could serve as a potential target to ameliorate aging-associated intestinal pathologies.
CD133 is a wildly used cancer stem cell marker. The purpose of this study was to explore the significance of CD133 mRNA in human cancers mainly based on The Cancer Genome Atlas (TCGA) database. Bioinformatic analyses were done by using public repositories, including BioGPS, SAGE Genie tools, Oncomine analysis, Regulome Explorer, COSMIC analysis, and Kaplan-Meier Plotter. The main findings in this study were: 1) High CD133 mRNA was correlated with a benign survival rate of gastric cancer and lung cancer; 2) Transmembrane protein 125 (TMEM125) in bladder urothelial carcinoma and intercellular adhesion molecule 2 (ICAM2) in ovarian serous cystadenocarcinoma were closely related to CD133 expression; 3) The location and the topological structure of CD133 protein were not determined by its transcript variant in cancer cells; 4) CD38 and CD200 may be used as novel surface markers for solid cancers. However, the mechanism of these findings is not completely clear, further studies have to be performed in the future.
Please note the following errors in the original version. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.
The PTFE micro-bioreactor promotes 3D cell rearrangement and maintains high plasticity in epigenetically erased Oct4-GFP murine fibroblasts. (a) Cells encapsulated in PTFE and exposed to 5-aza-CR for 18 h formed 3D spherical structures, that were stably maintained for the entire length of the experiments (Scale bar, 100 μm). (b) Fibroblasts became GFP positive, indicating the onset of Oct4 gene expression (Scale bar, 100 μm). (c) Gene expression profiling of pluripotency-related genes (Oct4, Nanog, Rex1, and Sox2) in untreated fibroblasts (T0), fibroblasts exposed to 5-aza-CR (Post 5-aza-CR) and at different time points of culture. Gene expression levels are reported with highest expression set to 1 and all other times relative to this. Different superscripts denote significant differences (P < 0.05)
The PTFE micro-bioreactor promotes 3D cell rearrangement and induces ultrastructural modifications in epigenetically erased human fibroblasts. (a) After 5-aza-CR incubation, fibroblasts plated on plastic dishes (Group A) changed their typical elongated shape into a round epithelioid aspect and retained a monolayer distribution. Cell size was smaller, and nuclei became larger and granular. (Scale bar, 100 μm). (b) Cells encapsulated in PTFE (Group B) and treated with the demethylating agent formed 3D spherical structures, that were stably maintained for the entire length of the experiments (Scale bar, 200 μm). (C) Cells maintained a normal karyotype. (d-e) Untreated fibroblasts displayed a spindle shaped morphology. (f-g) Cells encapsulated in PTFE and subjected to 5-aza-CR treatment became ovoidal with autophagic phenomena. (h-i) They showed a roundish shape, high nucleus to cytoplasm ratio, nuclei with euchromatin and large reticulated nucleoli, few organelles, and large intercellular spaces for the entire length of the experiments
The PTFE micro-bioreactor enhances the demethylating effect of 5-aza-CR, boosts pluripotency gene transcription and maintains long-term high plasticity in epigenetically erased human fibroblasts. (a) Global DNA methylation levels of cells plated on standard plastic dishes (Group A) or encapsulated in PTFE (Group B), exposed to 5-aza-CR (Post 5-aza-CR) and cultured in ESC medium. Bars represent the mean ± SD of three independent experiments with five independent biological replicates. Different superscripts denote significant differences (P < 0.05). (b) Gene expression changes in epigenetically erased fibroblasts plated on standard plastic dishes (Group A) or encapsulated in PTFE (Group B). Expression pattern of pluripotency-related genes (OCT4, NANOG, REX1, and SOX2), ten-eleven translocation family member TET2, MET markers (EPCAM, CDH1) and fibroblast specific marker (THY1). Gene expression levels are shown for untreated fibroblasts (T0), fibroblasts exposed to 5-aza-CR (Post 5-aza-CR), and at different time points of culture. Values are reported with highest expression set to 1 and all other times relative to this. Different superscripts denote significant differences (P < 0.05)
Activation of the Hippo signaling pathway in PTFE encapsulated cells. (a) Western blots for TAZ and SMAD2 proteins in epigenetically erased fibroblasts, plated on standard plastic dishes (Group A). The densitometry results (arbitrary units) of the Western blots are shown as a bar graph. Bars represent the mean ± SD of three independent experiments with five independent biological replicates. Different superscripts denote significant differences (P < 0.05). Representative Western blots for each protein are also shown. (b) Western blots for TAZ and SMAD2 proteins in epigenetically erased fibroblasts, encapsulated in PTFE (Group B). The densitometry results (arbitrary units) of the Western blots are shown as a bar graph. Bars represent the mean ± SD of three independent experiments with five independent biological replicates. Different superscripts denote significant differences (P < 0.05). Representative Western blots for each protein are also shown. (c) Western blots for TAZ and SMAD2 proteins in PTFE encapsulated cells at day 28 of culture, after TAZ siRNA transfection (28d + siTAZ). The densitometry results (arbitrary units) of the Western blots are shown as a bar graph. Bars represent the mean ± SD of three independent experiments with five independent biological replicates. Different superscripts denote significant differences (P < 0.05). Representative Western blots for each protein are also shown. (d) Pluripotency-related gene (OCT4, NANOG, REX1, and SOX2) expression levels in Group B cells at day 28 of culture (28d) and after TAZ siRNA transfection (28d + siTAZ). Values are reported with highest expression set to 1 and all other times relative to this. Different superscripts denote significant differences (P < 0.05)
Phenotype definition is driven by epigenetic mechanisms as well as directly influenced by the cell microenvironment and by biophysical signals deriving from the extracellular matrix. The possibility to interact with the epigenetic signature of an adult mature cell, reversing its differentiated state and inducing a short transient high plasticity window, was previously demonstrated. In parallel, in vitro studies have shown that 3D culture systems, mimicking cell native tissue, exert significant effects on cell behavior and functions. Here we report the production of “PTFE micro-bioreactors” for long-term culture of epigenetically derived high plasticity cells. The system promotes 3D cell rearrangement, global DNA demethylation and elevated transcription of pluripotency markers, that is dependent on WW domain containing transcription regulator 1 (TAZ) nuclear accumulation and SMAD family member 2 (SMAD2) co-shuttling. Our findings demonstrate that the use of 3D culture strategies greatly improves the induction and maintenance of a high plasticity state.
Antigenic profiles used to identify endothelial progenitor cells by flow cytometry in studies addressing “endothelial progenitor cells and cardiovascular risk” or “endothelial progenitor cells and acute coronary syndromes” as electronic search keywords. This graph represents the frequency distributions of the 19 different antigenic profiles used to quantify EPCs in 17 published studies that established a relationship between the possible antigenic profiles of EPCs and cardiovascular diseases (coronary artery disease, acute coronary syndromes and acute myocardial infarction)
Cell culture assays for endothelial progenitor cells. This illustration displays the different culture assays described in the literature for EPCs, namely early EPCs, CFU-EC/CFU-Hill and ECFCs/late EPCs. Adapted from Fadini et al. (2012). CD, cluster of differentiation; CFU-EC, colony forming unit of endothelial cell; Dil-acLDL, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-acetylated low density lipoproteins; ECFC, endothelial colony forming cell; EPC, endothelial progenitor cell; KDR, kinase insert domain receptor; MNCs, mononuclear cells; vWF, von Willebrand factor
Endothelial Progenitor Cells pathway after acute myocardial infarction. The rupture of an atherosclerotic plaque triggers a series of inflammatory pathways and the obstruction of blood flow through the artery with a resulting reduced oxygen supply (hypoxia) to the heart and, consequently, ischemia of the heart muscle, which may lead to acute myocardial infarction (AMI). After AMI, EPCs disengage from the bone marrow (with involvement of MMP-9, SDF-1, VEGF and SDF-1/CXCR4 axis) and are mobilized following cytokine secretion (HIF-1, SDF-1, VEGF, IL-1β, IL-8, among others). They home into the damaged blood vessels supplying the ischemic myocardium and incorporate into new blood vessels, repairing the ischemic tissue through vasculogenesis. CXCR4, C-X-C chemokine receptor type 4 (CXCR4); EPCs, endothelial progenitor cells; HIF-1, hypoxia-inducible factor-1; IL-1β, interleukin-1β; IL-8, interleukin-8; MMP-9, matrix metalloproteinase-9; SDF-1, stromal cell-derived factor-1; VEGF, vascular endothelial growth factor
Bone marrow-derived endothelial progenitor cells (EPCs) play a key role in the maintenance of endothelial homeostasis and endothelial repair at areas of vascular damage. The quantification of EPCs in peripheral blood by flow cytometry is a strategy to assess this reparative capacity. The number of circulating EPCs is inversely correlated with the number of cardiovascular risk factors and to the occurrence of cardiovascular events. Therefore, monitoring EPCs levels may provide an accurate assessment of susceptibility to cardiovascular injury, greatly improving risk stratification of patients with high cardiovascular risk, such as those with an acute myocardial infarction. However, there are many issues in the field of EPC identification and quantification that remain unsolved. In fact, there have been conflicting protocols used to the phenotypic identification of EPCs and there is still no consensual immunophenotypical profile that corresponds exactly to EPCs. In this paper we aim to give an overview on EPCs-mediated vascular repair with special focus on acute coronary syndromes and to discuss the different phenotypic profiles that have been used to identify and quantify circulating EPCs in several clinical studies. Finally, we will synthesize evidence on the prognostic role of EPCs in patients with high cardiovascular risk.
Key DCM model requirements including relevant readouts (a) Insulin resistance – demonstrated by reduced insulin signaling, for example reduced Akt phosphorylation. (b) Metabolic shift – demonstrated by reduced glucose oxidation and increased FA oxidation. (c) Lipotoxicity – demonstrated by increased intracellular lipid accumulation and/or increased lipid peroxidation. (d) Hypertrophy – demonstrated by hypertrophic morphology and/or increased expression of hypertrophic markers such as BNP. (E) Altered functionality – demonstrated by impaired Ca²⁺-transient or contractility and/or mitochondrial dysfunction
iPSC derived diabetic cardiomyopathy model (a) Model setup: CMs derived from CDI-MRB iPSCs from Cellular Dynamics International were used in an induction medium protocol. Cardiac maintenance medium (CM): DMEM no glucose, 10mMHEPES, 2mML-carnitine, 5 mM creatine, 5 mM taurine, 1 mM ITS, 1 mM nonessential amino acids, linoleic-oleic acid (1xFFA) supplemented with 10 mM glucose. Maturation medium (MM) consisting of CM supplemented with 1xFFA. Diabetic medium (DM) consisting of CM supplemented with 20 mM glucose, 50uM palmitate (conjugated by 0.8%FAF-BSA), 15 mg/100 ml uric acid, 2xFFA and 10 nM endothelin-1 after day 10. The effect of the diabetic induction protocol on (b) Akt(S473)-phosphorylation, (c) Relative gene expression of hypertrophy- and substrate utilization markers and (d) Cardiomyocyte respiration - oxygen consumption rate measured by Seahorse XF in culture medium before and after oligomycin, 2,4-dinitrophenol and rotenone/antimycin A treatment. Error bars represent standard deviation and * indicates p < 0.05 compared to the control by non-parametric two-tailed Mann-Whitney Test
Development of cardiac insulin resistance Increased levels of circulating FA will trigger an increase in FA uptake and oxidation, which in turn will suppress glucose oxidation. To counteract rising blood glucose levels, more insulin will be secreted by the pancreas resulting in increasing blood insulin levels. As the cardiac insulin sensitivity is reduced, glucose uptake is further diminished and cells will become further reliant on FA for energy metabolism. As this feedback loop continues the resulting negative spiral will ultimately result in a state of insulin resistance and metabolic rigidity
The global burden of diabetes has drastically increased over the past decades and in 2017 approximately 4 million deaths were caused by diabetes and cardiovascular complications. Diabetic cardiomyopathy is a common complication of diabetes with early manifestations of diastolic dysfunction and left ventricular hypertrophy with subsequent progression to systolic dysfunction and ultimately heart failure. An in vitro model accurately recapitulating key processes of diabetic cardiomyopathy would provide a useful tool for investigations of underlying disease mechanisms to further our understanding of the disease and thereby potentially advance treatment strategies for patients. With their proliferative capacity and differentiation potential, human induced pluripotent stem cells (iPSCs) represent an appealing cell source for such a model system and cardiomyocytes derived from induced pluripotent stem cells have been used to establish other cardiovascular related disease models. Here we review recently made advances and discuss challenges still to be overcome with regard to diabetic cardiomyopathy models, with a special focus on iPSC-based systems. Recent publications as well as preliminary data presented here demonstrate the feasibility of generating cardiomyocytes with a diabetic phenotype, displaying insulin resistance, impaired calcium handling and hypertrophy. However, capturing the full metabolic- and functional phenotype of the diabetic cardiomyocyte remains to be accomplished. Electronic supplementary material The online version of this article (10.1007/s12015-018-9858-1) contains supplementary material, which is available to authorized users.
PFM-captured HAP stem cell colonies were used for implantation to the severed spinal cord in nude (allo transplantation) and immunocompetent mice (autologous transplantation). Experiment I: GFP-expressing HAP stem cell colonies from GFP-mouse hair follicles captured on PFM were implanted in the severed thoracic spinal cord of nude mice. Experiment II: HAP stem cell colonies from C57BL/6 J mice captured on PFM were implanted in the severed thoracic spinal cord of C57BL/6 J mice
HAP stem cell colonies differentiated into neurons and glial cells after culture for 5 days on PFM (a1, a2). Red = βIII tubulin (left) or GFAP (right); Blue = DAPI. Differentiated cells from HAP stem cell colonies contained 24.2% neurons (b1) and 72.8% glial cells (b2)
Seven weeks after implantation, the severed thoracic spinal cord of the implanted nude mouse was directly observed by fluorescence microscopy which showed that GFP-expressing HAP stem cells extended from the PFM and joined the severed thoracic spinal cord (a). Left panel = low magnification. Right panel = high-magnification of white boxed area. Immunofluorescence staining also showed that the nerves joined and HAP stem cells differentiated into neurons (b) and glial cells (c). Red = βIII tubulin (b) or GFAP (c); Green = GFP; Blue = DAPI. Left panels = low magnification. Bar = 100 μm. Right panels = high magnification of white boxed area. Bar = 100 μm
Implantation of HAP stem cell colonies encapsulated in PFM in C57BL/6J mice rejoined the severed thoracic spinal cord. H&E staining of the severed part of thoracic spinal cord in implanted mice (a). Immunostaining images of paraffin sections, 7 weeks after implantation of HAP stem cell colonies captured on PFM in the severed thoracic spinal cord fragments, showed that the HAP stem cells differentiated into neurons (b), and glial cells (c). Mice implanted with empty PFM and mice without implantation did not rejoin the thoracic spinal cord and showed granulation tissue in the severed part of the thoracic spinal cord (d, g). The severed part of the thoracic spinal cord did not differentiate into neurons (e, h) and glial cells (f, i). Upper panels = low magnification. Bar = 500 μm. Lower panels = high magnification of the white boxed area. Bar = 500 μm
HAP stem cell colonies encapsulated in PFM effected recovery of motor function. The BMS score of the mice implanted with encapsulated HAP stem cells showed a significant improvement compared with mice without implantation after four weeks. *P < 0.05; compared with empty PFM after five weeks. **P < 0.05. We determined the BMS score by observing the left leg of mice. n = 13 mice implanted HAP stem cells; n = 9 mice of empty PFM; n = 12 mice without implantation
Our previous studies showed that nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells, which reside in the bulge area of the hair follicle, could restore injured nerve and spinal cord and differentiate into cardiac muscle cells. Here we transplanted mouse green fluorescent protein (GFP)-expressing HAP stem-cell colonies enclosed on polyvinylidene fluoride membranes (PFM) into the severed thoracic spinal cord of nude mice. After seven weeks of implantation, we found the differentiation of HAP stem cells into neurons and glial cells. Our results also showed that PFM-captured GFP-expressing HAP stem-cell colonies assisted complete reattachment of the thoracic spinal cord. Furthermore, our quantitative motor function analysis with the Basso Mouse Scale for Locomotion (BMS) score demonstrated a significant improvement in the implanted mice compared to non-implanted mice with a severed spinal cord. Our study also showed that it is easy to obtain HAP stem cells, they do not develop teratomas, and do not loose differentiation ability when cryopreserved. Collectively our results suggest that HAP stem cells could be a better source compared to induced pluripotent stem cells (iPS) or embryonic stem (ES) cells for regenerative medicine, specifically for spinal cord repair.
Top-cited authors
Ariff Bongso
  • National University of Singapore
Mohamed Abumaree
  • King Saud bin Abdulaziz University for Health Sciences
Bill Kalionis
  • Royal Women's Hospital in Victoria
Gauthaman Kalamegam
  • Saveetha University
Mohammed Al Jumah
  • King Saud bin Abdulaziz University for Health Sciences