[Show abstract][Hide abstract] ABSTRACT: Background
We previously reported that induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CM) manifest beat rate variability (BRV) resembling heart rate variability (HRV) in human sinoatrial node (SAN). We now hypothesized the BRV-HRV continuum originates in pacemaker cells.
To investigate whether cellular BRV is a source of HRV dynamics, we hypothesized three-levels of interaction among different cardiomyocyte entities: (1) single pacemaker cells, (2) networks of electrically coupled pacemaker cells and (3) in situ SAN.
We measured BRV/HRV properties in single pacemaker cells, iPSC-derived contracting embryoid bodies (EBs) and electrocardiograms from the same individual.
Pronounced BRV/HRV were present at all three levels. Coefficient of variance (COV) of inter-beat intervals (IBI) and Poincaré plot SD1 and SD2 in single cells were 20x > EBs (P<0.05) and in situ heart (the latter two were similar, P>0.05). We also compared BRV magnitude among single cells, small (~5-10 cells) and larger EBs (>10 cells): BRV indices progressively increased (P<0.05) as cell number decreased. Disrupting intracellular Ca2+ handling markedly augmented BRV magnitude, revealing a unique bi-modal firing pattern, suggesting intracellular mechanisms contribute to BRV/HRV and the fractal behavior of heart rhythm.
The decreased BRV magnitude in transitioning from single cell to EB suggests HRV of hearts in situ originates from summation and integration of multiple cell-based oscillators. Hence, complex interactions among multiple pacemaker cells and intracellular Ca2+ handling determine HRV in humans and isolated cardiomyocyte networks.
[Show abstract][Hide abstract] ABSTRACT: Isolated microvessel-residing pericytes and pericytes from human pluripotent stem cells (hPSCs) exhibit mesenchymal stem cell-like characteristics and therapeutic properties. Despite growing interest in pericyte-based stem cell therapy, their immunogenicity and immunomodulatory effects on nonactivated T cells are still poorly defined, in particular those of vasculogenic hPSC pericytes. We found that tissue-embedded and unstimulated cultured hPSC- or tissue-derived pericytes constitutively expressed major histocompatibility complex (MHC) class I and the inhibitory programmed cell death-ligand 1/2 (PD-L1/2) molecules but not MHC class II or CD80/CD86 costimulatory molecules. Pretreatment with inflammatory mediators failed to induce an antigen-presenting cell-like phenotype in stimulated pericytes. CD146(+) pericytes from hPSCs did not induce activation and proliferation of allogeneic resting T cells independent of interferon (IFN)-γ prestimulation, similarly to pericytes from human brain or placenta. Instead, pericytes mediated a significant increase in the frequency of allogeneic CD25(high)FoxP3(+) regulatory T cells when cocultured with nonactivated peripheral blood T cells. Furthermore, when peripheral blood CD25(high) regulatory T cells (Tregs) were depleted from isolated CD3(+) T cells, pericytes preferentially induced de novo formation of CD4(+)CD25(high)FoxP3(+)CD127(-), suppressive regulatory T cells. Constitutive expression of PD-L1/2 and secretion of transforming growth factor-β by hPSC pericytes directly regulated generation of pericyte-induced Tregs. Pericytes cotransplanted into immunodeficient mice with allogeneic CD25(-) T cells maintained a nonimmunogenic phenotype and mediated the development of functional regulatory T cells. Together, these findings reveal a novel feature of pericyte-mediated immunomodulation distinguished from immunosuppression, shared by native tissue pericytes and hPSC pericytes, and support the notion that pericytes can be applied for allogeneic cell therapy.
No preview · Article · Sep 2014 · STEM CELLS TRANSLATIONAL MEDICINE
[Show abstract][Hide abstract] ABSTRACT: Extracellular matrix (ECM) has been utilized as a biological scaffold for tissue engineering applications in a variety of body systems, due to its bioactivity and biocompatibility. In the current study we developed a modified protocol for the derivation of mesenchymal progenitor cells (MPCs) from human embryonic stem cells as well as human induced pluripotent stem cells originating from hair follicle keratinocytes (HFKTs). ECM was produced from these MPCs and characterized in compare to adipose mesenchymal stem cells (aMSCs) ECM. Exploiting an electrospun biodegradable nanofiber layer (NFL) which provide mechanical support, and an optimized decellularization treatment we were able to generate an available "off-the shelf" implantable product (NFL-ECM). Using rat subcutaneous transplantation model we demonstrate that this stem cell derived construct is biocompatible, biodegradable and provides great potential for tissue regeneration.
No preview · Article · Sep 2014 · Tissue Engineering Part A
[Show abstract][Hide abstract] ABSTRACT: To characterize, using 3D transperineal ultrasound, the appearance, position, and dimensions of mesh implants following minimally invasive abdominal sacrocolpopexy.
In women who underwent sacrocolpopexy, mesh was evaluated at rest and on maximal valsalva, on all 3D orthogonal planes and rendered views. Mesh dimensions were obtained by 3D processing in the midsagittal and coronal plane (anterior, posterior, sacral arm), and analyzed offline, blinded to clinical data.
Overall 62 women, mean age 58.4 years (range 42-79) were evaluated at 9 months mean (range 1-26) following surgery. The anterior arm of the mesh was caudal to the lowermost point of descent of the anterior compartment in 56 (90%), equally positioned in 5 (8.1%) and cranial in 1. The posterior arm was caudal in 44 (71%), equally positioned in 16 (25.8%) and cranial in 2 (3.2%). The Y connection and the sacral arm of the mesh could not be adequately seen due to ultrasound physics, due to large recurrent rectoceles, due to echogenic stools or from folding of mesh remnants. Folding of the mesh was seen in 46 (74.2%) women, of the anterior arm in 5 (8.1%) and of the posterior arm in 23 (37.1%). Folding occurred caudally in 26 (56.5%), proximally in 11 (23.9%) and in both areas in 9 (19.6%). There were no erosions.
Mesh visualization following minimally invasive abdominal sacrocolpopexy procedures using transperineal 3D/4D ultrasound is feasible. Studies are needed to evaluate the correlation between ultrasound measures and prolapse recurrence or mesh erosions.
No preview · Article · Apr 2014 · Ultrasound in Obstetrics and Gynecology
[Show abstract][Hide abstract] ABSTRACT: Adenosine to Inosine (A-to-I) RNA editing is a post-transcriptional, site specific modification process, catalyzed by Adenosine Deaminase Acting on RNA (ADAR) gene family members. Since ADARs act on double strand RNA, most A-to-I editing occurs within repetitive elements, particularly Alu elements, as the result of the inherent property of these sequences to fold and form double strands. A-to-I editing was recently implicated in the regulation of human embryonic stem cells (hESC). Spontaneous and neuronal differentiation of hESC was shown to result in a decrease in A-to-I editing levels. Knockdown of ADAR1 in hESCs results in elevation of the expression of differentiation related genes. In addition, we found that hESCs are not susceptible to ADAR1 overexpression. The current study shows that the editing levels of induced pluripotent stem cells (iPSCs) change throughout reprogramming, from a source cell level to a level similar to that of hESCs. Up or down regulation of the ADAR1 level in human foreskin fibroblast (HFF) cells prior to induction of reprogramming results in varied reprogramming efficiencies. Furthermore, HFF-iPSC early clones derived from source cells in which the ADAR1 level was downregulated, lose their iPSC properties shortly after iPSC colony formation and exhibit instead characteristics of cancer cells. Taken together, our results imply a role for A-to-I editing in the regulation of pluripotency induction as well as in the maintenance of early iPSC properties.
No preview · Article · Nov 2013 · Stem cells and development
[Show abstract][Hide abstract] ABSTRACT: Human induced pluripotent stem cells (hiPSCs) have great potential as a robust source of progenitors for regenerative medicine. The novel technology also enables the derivation of patient-specific cells for applications to personalized medicine, such as for personal drug screening and toxicology. However, the biological characteristics of iPSCs are not yet fully understood and their similarity to human embryonic stem cells (hESCs) is still unresolved. Variations among iPSCs, resulting from their original tissue or cell source, and from the experimental protocols used for their derivation, significantly affect epigenetic properties and differentiation potential. Here we review the potential of iPSCs for regenerative and personalized medicine, and assess their expression pattern, epigenetic memory and differentiation capabilities in relation to their parental tissue source. We also summarize the patient-specific iPSCs that have been derived for applications in biological research and drug discovery; and review risks that must be overcome in order to use iPSC technology for clinical applications.
No preview · Article · May 2013 · Current Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the "voltage-clock," where the hyperpolarization-activated funny current If causes diastolic depolarization that triggers action potential cycling; and (ii) the "Ca(2+) clock," where cyclical release of Ca(2+) from Ca(2+) stores depolarizes the membrane during diastole via activation of the Na(+)-Ca(2+) exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current- and voltage-clamp recordings from the same cell showed the so-called "voltage and Ca(2+) clock" pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the "voltage or Ca(2+) clock" produced a similar depolarization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, real-time PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca(2+)-activated intermediate K(+) conductance (IKCa, KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IKCa inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IKCa appears to play a crucial role in human embryonic cardiac automaticity.
Full-text · Article · Apr 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Maternal infection or inflammation may induce fetal inflammatory responses associated with fetal injury and cerebral palsy. We sought to assess the inflammation-associated neuroprotective potential of prophylactic N-acetyl-cysteine (NAC). We examined the effect of NAC on prevention of maternal lipopolysaccharide (LPS)-induced neonatal brain injury using magnetic resonance imaging.
Pregnant Sprague Dawley dams (n = 5-8) at embryonic day 18 received intraperitoneal injection of LPS or saline at time 0. Animals were randomized to receive 2 intravenous injections of NAC or saline (time -30 and 120 minutes). Pups were delivered spontaneously and allowed to mature until postnatal day 25. Female offspring were examined by magnetic resonance brain imaging and analyzed using voxel-based analysis after spatial normalization. T2 relaxation time was used to assess white matter injury and diffusion tensor imaging for apparent diffusion coefficient (ADC) to assess white and gray matter injury.
Offspring of LPS-treated dams exhibited significantly increased T2 levels and increased ADC levels in white and gray matter (eg, hypothalamus, motor cortex, corpus callosum, thalamus, hippocampus), consistent with diffuse cerebral injury. In contrast, offspring of NAC-treated LPS dams demonstrated similar T2 and ADC levels as control in both white and gray matter.
Maternal NAC treatment significantly reduced evidence of neonatal brain injury associated with maternal LPS. These studies suggest that maternal NAC therapy may be effective in human deliveries associated with maternal/fetal inflammation.
No preview · Article · Mar 2013 · American journal of obstetrics and gynecology
[Show abstract][Hide abstract] ABSTRACT: The Silences of the Archives, the Reknown of the Story.
The Martin Guerre affair has been told many times since Jean de Coras and Guillaume Lesueur published their stories in 1561. It is in many ways a perfect intrigue with uncanny resemblance, persuasive deception and a surprizing end when the two Martin stood face to face, memory to memory, before captivated judges and a guilty feeling Bertrande de Rols. The historian wanted to go beyond the known story in order to discover the world of the heroes. This research led to disappointments and surprizes as documents were discovered concerning the environment of Artigat’s inhabitants and bearing directly on the main characters thanks to notarial contracts. Along the way, study of the works of Coras and Lesueur took a new direction. Coming back to the affair a quarter century later did not result in finding new documents (some are perhaps still buried in Spanish archives), but by going back over her tracks, the historian could only be struck by the silences of the archives that refuse to reveal their secrets and, at the same time, by the possible openings they suggest, by the intuition that almost invisible threads link here and there characters and events.
Full-text · Article · Feb 2013 · Heart rhythm: the official journal of the Heart Rhythm Society
[Show abstract][Hide abstract] ABSTRACT: During the process of human embryonic development, blood islands develop alongside the endoderm, which segregate into individual hemangioblasts that are surrounded by flattened endothelial precursor cells. The hemangioblasts mature into the first blood cells, while the endothelial precursors develop into blood vessel endothelium. New vascular formation, termed vasculogenesis, takes place within the embryo. The role of blood cell production is taken over by a series of embryonic organs, such as the liver, spleen, thymus, and bone marrow. The bipotential hemangioblast produces the primitive erythroid and endothelial progenitor cells, and the hemogenic endothelium gives rise to hematopoietic stem cells and endothelial progenitors. This chapter discusses the ability of these progenitors of hematopoiesis (blood cell repopulation), vasculogenesis or angiogenesis. The role of human embryonic stem cells as a source for vascular progenitors is also discussed. Human embryoid bodies (hEBs) are formed by the spontaneous differentiation of hESCs, and comprise multilineage tissues from endodermal, ectodermal, and mesodermal origin. Several experimental procedures have been developed to explore the endothelial potential of hESCs. Undifferentiated human embryonic stem cells (hESCs) form teratomas once injected into severe combined immunodeficient (SCID) mice. During teratoma formation from hESCs, two parallel vascular processes occur: angiogenesis of host vasculature into the forming human teratoma; and vasculogenesis of spontaneously differentiating hESCs. In further studies of human vasculogenesis, various vascular markers should be examined and evaluated in the course of spontaneous differentiation.
[Show abstract][Hide abstract] ABSTRACT: Maternal infection is associated with oxidative stress (OS) and inflammatory responses. We have previously shown that maternal exposure to lipopolysaccharide (LPS) at E18 alters the subsequent offspring immune response. As immune responses are mediated, in part, by OS, we sought to determine if maternal inflammation during pregnancy programs offspring OS and C-reactive protein (CRP) levels. Pregnant Sprague-Dawley rats received intraperitoneal (i.p.) injections of saline or LPS at 18 days’ gestation (n = 4), and pups delivered spontaneously at term. At postnatal day 24, male and female offspring received i.p. injection of LPS. Serum lipid peroxides formation (PD) and CRP levels were determined before and at 4 h following the LPS injection. Pups of LPS-exposed dams had significantly higher basal OS (PD 29.4 ± 5.4 v. 10.1 ± 4.8 nmol/ml) compared with controls. In response to LPS, CRP levels (20.4 ± 2.8 v. 5.7 ± 1.0 ng/ml) were significantly higher among pups of LPS-exposed dams than controls. Prenatal maternal exposure to LPS increases baseline OS levels in neonates and CRP levels in response to LPS. These results suggest that maternal inflammation during the antenatal period may induce long-term sequelae in the offspring that may predispose to adult disease.
[Show abstract][Hide abstract] ABSTRACT: Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2) and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC), as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1-p110 isoform. We were unable to achieve overexpression of ADAR1-p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24-48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms underlying the immunomodulatory effects of mesenchymal stem cells (MSCs) have been essentially studied in conditions of strong T cell activation that represents extreme situation and induces rapid death of activated lymphocytes. The objective of this study was to investigate these mechanisms in absence of additional polyclonal activation. In cocultures of peripheral mononuclear blood cells with hMSC, we observed a striking decreased expression of CD8 level on CD8+ cells, together with decreased CD28 and CD44 expression and impaired IFN-gamma and Granzyme B production. This effect was specific to hMSCs, since it was not observed with several other cell lines. Down-regulation of CD8 expression required CD14+ monocytes in direct contact with the CD8+ cells, while the effects of hMSCs on the CD14+ cells were essentially mediated by soluble factors. The CD14+ monocytes exhibited a tolerogenic pattern when co-cultured with hMSCs, with a clear decrease in CD80 and CD86 co-stimulatory molecules, and an increase in the inhibitory receptors ILT-3 and ILT-4. MSC-preconditioned CD8+ cells had similar effects on monocytes and were able to inhibit lymphocyte proliferation. Injection of human MSCs in humanized NSG mice showed similar trends, in particular decreased CD44 and CD28 on human immune cells. Altogether, our study demonstrates a new immunomodulation mechanism of action of hMSCs through the modulation of CD8+ cells towards a non-cytotoxic/suppressive phenotype. This mechanism of action has to be taken into account in clinical trials, where it should be beneficial in grafts and autoimmune diseases, but potentially detrimental in malignant diseases.