Kazim H. Narsinh

Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising candidate cell sources for regenerative medicine. However, despite the common ability of hiPSCs and hESCs to differentiate into all 3 germ layers, their functional equivalence at the single cell level remains to be demonstrated. Moreover, single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here, we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs, suggesting that hiPSCs occupy an alternate, less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exercised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs, particularly when producing differentiated cells for regenerative medicine aims.

Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors

Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ∼4 weeks.

Rosuvastatin enhances the therapeutic efficacy of adipose-derived mesenchymal stem cells for myocardial infarction via PI3K/Akt and MEK/ERK pathways

The poor viability of transplanted stem cells hampers their therapeutic efficacy for treatment of myocardial infarction. The aim of this study was to investigate whether rosuvastatin improved survival of adipose-derived mesenchymal stem cells (AD-MSCs) after transplantation into infarcted hearts. AD-MSCs isolated from Tg(Fluc-egfp) mice which constitutively express both firefly luciferase (Fluc) and enhanced green fluorescent protein were transplanted into infarcted hearts with or without rosuvastatin administration. Longitudinal in vivo bioluminescence imaging and histological staining revealed that rosuvastatin enhanced the survival of engrafted AD-MSCs. Furthermore, combined therapy of AD-MSC and rosuvastatin reduced fibrosis, decreased cardiomyocyte apoptosis, and preserved heart function. AD-MSCs were then subjected to hypoxia and serum deprivation injury in vitro to mimic the ischemic environment. Rosuvastatin (10−6 mmol/L) enhanced the viability and paracrine effect of AD-MSCs, and decreased their apoptotic rate. Western blotting revealed that rosuvastatin supplementation increased Akt and ERK phosphorylation, which resulted in FoxO3a phosphorylation and nuclear export. In addition, rosuvastatin administration decreased the pro-apoptotic proteins Bim and Bax, and increased the anti-apoptotic proteins Bcl-xL and Bcl-2. Furthermore, these effects were abolished by PI3K inhibitor LY294002 and MEK1/2 inhibitor U0126. This study demonstrates that rosuvastatin may improve the survival of engrafted AD-MSCs at least in part through the PI3K/Akt and MEK/ERK1/2 signaling pathways. Combination therapy with rosuvastatin and AD-MSCs has a synergetic effect on improving myocardial function after infarction.

The poly(A)-dependent transcriptional pause is mediated by CPSF acting on the body of the polymerase

Eukaryotic poly(A) signals direct mRNA 3′-end processing and also pausing and termination of transcription. We show that pausing and termination require the processing factor CPSF, which binds the AAUAAA hexamer of the mammalian poly(A) signal. Pausing does not require the RNA polymerase II C-terminal domain (CTD) or the cleavage stimulation factor, CstF, that binds the CTD. Pull-down experiments show that CPSF binds, principally through its 30-kDa subunit, to the body of the polymerase. CPSF can also bind CstF, but this seems to be mutually exclusive with polymerase binding. We suggest that CPSF, while binding the body of the polymerase, scans for hexamers in the extruding RNA. Any encounter with a hexamer triggers pausing. If the hexamer is part of a functional poly(A) signal, CstF is recruited and binds CPSF, causing it to release the polymerase body and move (with CstF) to the CTD.

Derivation of Human Induced Pluripotent Stem Cells for Cardiovascular Disease Modeling

The successful derivation of human induced pluripotent stem cells (hiPSCs) by dedifferentiation of somatic cells offers significant potential to overcome obstacles in the field of cardiovascular disease. hiPSC derivatives offer incredible potential for new disease models and regenerative medicine therapies. However, many questions remain regarding the optimal starting materials and methods to enable safe, efficient derivation of hiPSCs suitable for clinical applications. Initial reprogramming experiments were carried out using lentiviral or retroviral gene delivery methods. More recently, various nonviral methods that avoid permanent and random transgene insertion have emerged as alternatives. These include transient DNA transfection using plasmids or minicircles, protein transduction, or RNA transfection. In addition, several small molecules have been found to significantly augment hiPSC derivation efficiency, allowing the use of a fewer number of genes during pluripotency induction. We review these various methods for the derivation of hiPSCs, focusing on their ultimate clinical applicability, with an emphasis on their potential for use as cardiovascular therapies and disease-modeling platforms.

Comparison of Human Induced Pluripotent and Embryonic Stem Cells: Fraternal or Identical Twins?

Human induced pluripotent stem cells (hiPSCs) have been hailed as an effective replacement for human embryonic stem cells (hESCs) and a prime candidate cell source for regenerative medicine aims. Both hESCs and hiPSCs share the important properties of self-renewal and pluripotency; that is, they are theoretically capable of generating unlimited amounts of any differentiated cell in the human body. However, accumulating reports of gene expression differences between hESCs and hiPSCs have led many to question the equivalence of these two promising cell types. Seemingly random variation in the differentiation propensity of hiPSCs to neural,1 cardiovascular,2 and hemangioblastic lineages3 has frustrated investigators hoping to better exploit their potential for disease modeling and cell replacement therapies. In light of these somewhat dispiriting results, the recent publication of genome-wide reference “scorecards” for monitoring the quality and utility of 32 human pluripotent stem cell lines is a welcome advance.4 Such advances are crucial to aiding our ability to predict a cell line’s differentiation propensity in a high-throughput fashion.

Preclinical derivation and imaging of autologously transplanted canine induced pluripotent stem cells

Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.

Early stem cell engraftment predicts late cardiac functional recovery: preclinical insights from molecular imaging.

Background— Human cardiac progenitor cells have demonstrated great potential for myocardial repair in small and large animals, but robust methods for longitudinal assessment of their engraftment in humans is not yet readily available. In this study, we sought to optimize and evaluate the use of positron emission tomography (PET) reporter gene imaging for monitoring human cardiac progenitor cell (hCPC) transplantation in a mouse model of myocardial infarction. Methods and Results— hCPCs were isolated and expanded from human myocardial samples and stably transduced with herpes simplex virus thymidine kinase (TK) PET reporter gene. Thymidine kinase-expressing hCPCs were characterized in vitro and transplanted into murine myocardial infarction models (n=57). Cardiac echocardiographic, magnetic resonance imaging and pressure-volume loop analyses revealed improvement in left ventricular contractile function 2 weeks after transplant (hCPC versus phosphate-buffered saline, P<0.03). Noninvasive PET imaging was used to track hCPC fate over a 4-week time period, demonstrating a substantial decline in surviving cells. Importantly, early cell engraftment as assessed by PET was found to predict subsequent functional improvement, implying a “dose–effect” relationship. We isolated the transplanted cells from recipient myocardium by laser capture microdissection for in vivo transcriptome analysis. Our results provide direct evidence that hCPCs augment cardiac function after their transplantation into ischemic myocardium through paracrine secretion of growth factors. Conclusions— PET reporter gene imaging can provide important diagnostic and prognostic information regarding the ultimate success of hCPC treatment for myocardial infarction.

In Vivo Functional and Transcriptional Profiling of Bone Marrow Stem Cells After Transplantation Into Ischemic Myocardium

Objective— Clinical trials of bone marrow–derived stem cell therapy for the heart have yielded variable results. The basic mechanism(s) that underlies their potential efficacy remains unknown. In the present study, we evaluated the survival kinetics, transcriptional response, and functional outcome of intramyocardial bone marrow mononuclear cell (BMMC) transplantation for cardiac repair in a murine myocardial infarction model. Methods and Results— We used bioluminescence imaging and high-throughput transcriptional profiling to evaluate the in vivo survival kinetics and gene expression changes of transplanted BMMCs after their engraftment into ischemic myocardium. Our results demonstrate short-lived survival of cells following transplant, with less than 1% of cells surviving by 6 weeks posttransplantation. Moreover, transcriptomic analysis of BMMCs revealed nonspecific upregulation of various cell regulatory genes, with a marked downregulation of cell differentiation and maturation pathways. BMMC therapy caused limited improvement of heart function as assessed by echocardiography, invasive hemodynamics, and positron emission tomography. Histological evaluation of cell fate further confirmed findings of the in vivo cell tracking and transcriptomic analysis. Conclusion— Collectively, these data suggest that BMMC therapy, in its present iteration, may be less efficacious than once thought. Additional refinement of existing cell delivery protocols should be considered to induce better therapeutic efficacy.

Comparison of Gene-Transfer Efficiency in Human Embryonic Stem Cells

Technologies designed to allow manipulation and modification of human embryonic stem (hES) cells are numerous and vary in the complexity of their methods, efficiency, reliability, and safety. The most commonly studied and practiced of these methods include electroporation, lipofection, nucleofection, and lentiviral transduction. However, at present, it is unclear which protocol offers the most efficient and reliable method of gene transfer to hES cells. In this study, a bi-fusion construct with ubiquitin promoter driving enhanced green fluorescent protein reporter and the firefly luciferase (pUb-eGFP-Fluc) along with neomycin selection marker was used for in vitro and in vivo studies. In vitro studies examined the transfection efficiency and viability of each technique using two hES cell lines (male H1 and female H9 cells). Lentiviral transduction demonstrated the highest efficiency (H1: 25.3 ± 4.8%; H9: 22.4 ± 6.5%) with >95% cell viability. Nucleofection demonstrated transfection efficiency of 16.1 ± 3.6% (H1) and 5.8 ± 3.2% (H9). However, minimal transfection efficiency was observed with electroporation (2.1 ± 0.4% (H1) and 1.9 ± 0.3% (H9)) and lipofection (1.5 ± 0.5% (H1) and 1.3 ± 0.2% (H9); P < 0.05 vs. lentiviral transduction). Electroporation also demonstrated the highest cell death (62 ± 11% (H1) and 42 ± 10% (H9)) followed by nucleofection (25 ± 9% (H1) and 30 ± 15 (H9)). Importantly, lentiviral transduction generated a greater number of hES cell lines stably expressing the double-fusion reporter gene (hES-DF) compared to other transfection techniques. Finally, following subcutaneous transplantation into immunodeficient nude mice, the hES-eGFP-Fluc cells showed robust proliferation as determined by longitudinal bioluminescence imaging. In summary, this study demonstrates that lentiviral transduction and nucleofection are efficient, simple, and safe techniques for reliable gene transfer in hES cells. The double-fusion construct provides an attractive approach for generating stable hES cell lines and monitoring engraftment and proliferation in vitro and in vivo.