Differentiation Of Human iPSCs Research Articles

Electrospun biomaterial scaffolds with varied topographies for neuronal differentiation of human-induced pluripotent stem cells.

In this study, we investigated the effect of micro and nanoscale scaffold topography on promoting neuronal differentiation of human induced pluripotent stem cells (iPSCs) and directing the resulting neuronal outgrowth in an organized manner. We used melt electrospinning to fabricate poly (ε-caprolactone) (PCL) scaffolds with loop mesh and biaxial aligned microscale topographies. Biaxial aligned microscale scaffolds were further functionalized with retinoic acid releasing PCL nanofibers using solution electrospinning. These scaffolds were then seeded with neural progenitors derived from human iPSCs. We found that smaller diameter loop mesh scaffolds (43.7 ± 3.9 µm) induced higher expression of the neural markers Nestin and Pax6 compared to thicker diameter loop mesh scaffolds (85 ± 4 µm). The loop mesh and biaxial aligned scaffolds guided the neurite outgrowth of human iPSCs along the topographical features with the maximum neurite length of these cells being longer on the biaxial aligned scaffolds. Finally, our novel bimodal scaffolds also supported the neuronal differentiation of human iPSCs as they presented both physical and chemical cues to these cells, encouraging their differentiation. These results give insight into how physical and chemical cues can be used to engineer neural tissue.

Enhanced Lung Epithelial Specification of Human Induced Pluripotent Stem Cells on Decellularized Lung Matrix

Whole-lung scaffolds can be created by perfusion decellularization of cadaveric donor lungs. The resulting matrices can then be recellularized to regenerate functional organs. This study evaluated the capacity of acellular lung scaffolds to support recellularization with lung progenitors derived from human induced pluripotent stem cells (iPSCs). Whole rat and human lungs were decellularized by constant-pressure perfusion with 0.1% sodium dodecyl sulfate solution. Resulting lung scaffolds were cryosectioned into slices or left intact. Human iPSCs were differentiated to definitive endoderm, anteriorized to a foregut fate, and then ventralized to a population expressing NK2 homeobox 1 (Nkx2.1). Cells were seeded onto slices and whole lungs, which were maintained under constant perfusion biomimetic culture. Lineage specification was assessed by quantitative polymerase chain reaction and immunofluorescent staining. Regenerated left lungs were transplanted in an orthotopic position. Activin-A treatment, followed by transforming growth factor-β inhibition, induced differentiation ofhuman iPSCs to anterior foregut endoderm as confirmed by forkhead box protein A2 (FOXA2), SRY (Sex Determining Region Y)-Box 17 (SOX17), and SOX2 expression. Cells cultured on decellularized lung slices demonstrated proliferation and lineage commitment after5 days. Cells expressing Nkx2.1 were identified at 40%to 60% efficiency. Within whole-lung scaffolds andunder perfusion culture, cells further upregulated Nkx2.1 expression. After orthotopic transplantation, grafts were perfused and ventilated by host vasculature and airways. Decellularized lung matrix supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Whole-organ scaffolds and biomimetic culture enable coseeding of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Orthotopic transplantation may enable further invivo graft maturation.

206. A Minimal A2 Ubiquitous Chromatin Opening Element (UCOE) Effectively Prevents Transgene Silencing and Maintains Tissue-Specificity of a Myeloid-Specific Promoter During Hematopoietic Differentiation of Human iPSC

206. A Minimal A2 Ubiquitous Chromatin Opening Element (UCOE) Effectively Prevents Transgene Silencing and Maintains Tissue-Specificity of a Myeloid-Specific Promoter During Hematopoietic Differentiation of Human iPSC

Comparison of different protocols for neural differentiation of human induced pluripotent stem cells

Although embryonic stem cells (ESCs) have enormous potentials due to their pluripotency, their therapeutic use is limited by ethical, biological and safety issues. Compared to ESCs, induced pluripotent stem cells (iPSCs) can be obtained from mouse or human fibroblasts by reprogramming. Numerous studies have established many protocols for differentiation of human iPSCs (hiPSCs) into neural lineages. However, the low differentiation efficiency of such protocols motivates researchers to design new protocols for high yield differentiation. Herein, we compared neural differentiation potential of three induction media for conversion of hiPSCs into neural lineages. In this study, hiPSCs-derived embryoid bodies were plated on laminin coated dishes and were treated with three induction media including (1) bFGF, EGF (2) RA and (3) forskolin, IBMX. Immunofluorescence staining and quantitative real-time PCR (qPCR) analysis were used to detect the expression of neural genes and proteins. qPCR analysis showed that the expression of neural genes in differentiated hiPSCs in forskolin, IBMX supplemented media was significantly higher than undifferentiated cells and those in induction media containing bFGF, EGF or RA. In conclusion, our results indicated a successful establishment protocol with high efficiency for differentiation of hiPSCs into neural lineages.

In vitro neurogenesis: development and functional implications of iPSC technology.

Neurogenesis is the developmental process regulating cell proliferation of neural stem cells, determining their differentiation into glial and neuronal cells, and orchestrating their organization into finely regulated functional networks. Can this complex process be recapitulated in vitro using induced pluripotent stem cell (iPSC) technology? Can neurodevelopmental and neurodegenerative diseases be modeled using iPSCs? What is the potential of iPSC technology in neurobiology? What are the recent advances in the field of neurological diseases? Since the applications of iPSCs in neurobiology are based on the capacity to regulate in vitro differentiation of human iPSCs into different neuronal subtypes and glial cells, and the possibility of obtaining iPSC-derived neurons and glial cells is based on and hindered by our poor understanding of human embryonic development, we reviewed current knowledge on in vitro neural differentiation from a developmental and cellular biology perspective. We highlight the importance to further advance our understanding on the mechanisms controlling in vivo neurogenesis in order to efficiently guide neurogenesis in vitro for cell modeling and therapeutical applications of iPSCs technology.

Nanofiber-based polyethersulfone scaffold and efficient differentiation of human induced pluripotent stem cells into osteoblastic lineage

Human induced pluripotent stem cells (iPSCs) have been shown to have promising potential for regenerative medicine and tissue engineering applications. In the present study, osteogenic differentiation of human iPSCs was evaluated on polyethersulfone (PES) nanofibrous scaffold. According to the results, higher significant expressions of common osteogenic-related genes such as runx2, collagen type I, osteocalcin and osteonectin was observed in PES seeded human iPSCs compared with control. Alizarin red staining and alkaline phosphatase activity of differentiated iPSCs demonstrated significant osteoblastic differentiation potential of these cells. In this study biocompatibility of PES nanofibrous scaffold confirmed by flattened and spreading morphology of iPSCs under osteoblastic differentiation inductive culture. Taking together, nanofiber-based PES scaffold seeded iPSCs showed the highest capacity for differentiation into osteoblasts-like cells. These cells and PES scaffold were demonstrated to have great efficiency for treatment of bone damages and lesions.

Abstract 247: Expression and Activity of K ATP Channel in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells of Nondiabetic and Type II Diabetic Patients

Pharmacological preconditioning with volatile anesthetics (APC) protects the myocardium against ischemia/reperfusion injury via multiple pathways that include the cardiac sarcolemmal (sarc) KATP channel. However, APC is altered or absent in metabolic diseases such as diabetes. To investigate the underlying mechanisms we evaluated the expression and activity of cardiac sarcKATP channel in cardiomyocytes (CMs) differentiated from induced pluripotent stem cells of non-diabetic (N-iPSCs) and type II diabetic (DM-iPSCs) patients. Differentiation of human iPSCs into CMs was confirmed by positive immunostaining for cardiac-specific markers and expression of green fluorescent protein under transcriptional control of cardiac promoter myosin light chain-2v that was delivered by lentiviral vector. Immunocytochemical analysis of cardiac sarcKATP channel subunits was carried out with Anti-Kir6.2 and Anti-SUR2A antibodies. Images were captured using a laser-scanning confocal microscope. Single KATP channel activity was monitored from excised inside-out patches at membrane potentials +80 mV to -80 mV, in symmetrical 140 mM K+. The channel was closed by 1 µM glibenclamide and 2 mM ATP. Immunocytochemical analysis showed the presence and colocalization of Kir6.2 and SUR2A proteins in CMs derived from both N-iPSCs and DM-iPSCs. Interestingly, sarcolemmal expression of these subunits appeared lower in DM-iPSCs. Electrophysiological recordings demonstrated that at 5 µM ATP internal single sarcKATP channels were open more frequently in N-iPSCs (7 of 16 cultures) than in DM-iPSCs (3 of 16 cultures). When measured at +40 mV, the single channel current amplitude (2.2 pA) and conductance (55 pS) were typical for the cardiac sarcKATP channel in N-iPSCs. By contrast, the DM-iPSC channels flickered between the open and closed state, resulting in higher noise level and decreased current amplitude (1.6 pA). This is the first report of sarcKATP channel expression and activity in CMs differentiated from human N-iPSCs and DM-iPSCs. The study suggests that compromised APC in human diabetic heart may in part result from altered expression of sarcKATP channel subunits and altered channel kinetics.

Differentiation Potential of Human Induced Pluripotent Stem Cells (iPSCs) to Nucleus Pulposus-Like Cells in Vitro

Introduction Autologous or allogeneic cell delivery to the herniated or degenerated intervertebral disk may promote tissue regeneration and arrest degeneration1–4. Induced pluripotent stem cells (iPSCs) derived from the patient's somatic cells represent an attractive cell source, having the potential to differentiate into various cell types5. To date, no studies have demonstrated that human iPSCs can differentiate into nucleus pulposus (NP) cells. We have previously shown that both human umbilical cord mesenchymal stromal cells and mouse iPSCs were able to display many NP-like cell markers after cultured in a laminin-rich 3D culture system for inducing differentiation6,7. The goal of this study is to evaluate whether human iPSCs cultured in this similar laminin-rich environment can also express a unique NP-like cell phenotype. Materials and Methods Cell Generation and Characterization iPSCs were generated from human embryonic dermal fibroblasts through transient inducible over-expression of transcription factors (OCT4, SOX2, KLF4 and MYC) by a lentiviral-based gene delivery system (a poly-cystronic vector with the Reverse Tetracycline Transactivator (M2rtTA) and doxycycline). For the derived colonies, immuno-cytochemistry was performed using a pluripotent markers kit (OCT4, SOX2, SSEA-4, TRA1-60, TRA1-81 and alkaline phosphatase, Applied StemCell). Selected iPSC colonies were maintained in culture upon a PMEF feeder layer. Cell Differentiation Undifferentiated iPSCs were seeded (106/well, Transwell) on wells precoated with Matrigel to generate a soft laminin-rich matrix6,7. Cells were cultured in conditioned medium collected from porcine NP tissues (DMEM + 1% ITS)8 for up to 28 days. Flow Cytometry Analysis Undifferentiated iPSCs were incubated with the antibodies against CD24, CD31, CD90, integrin subunits a3 (CD49c), a6 (CD49f), and b1 (CD29). For intracellular expression of transcription factors (Brachyury, OCT4), cells were permeabilized before incubation with primary antibodies. Cells were analyzed for fluorescence (Accuri C6) to quantify the percent of positively labeled cells. Immunohistochemistry Differentiated cells were harvested for cryo-sectioning. Cell morphology and proteoglycan synthesis was assessed by histological staining (H&E and Safranin O). Expression of NP markers was evaluated by immunostaining for matrix proteins (type II collagen (COL II), laminin 10 (LM511)), laminin-related receptors (CD239, integrin subunits a3, a6, b1, b4), and other markers (vimentin (VIM), cytokeratin 8 (KRT8), N-cadherin, CD24), as well as non-NP-markers (type I collagen, E-cadherin). Results iPSCs proliferated and formed colonies on PMEF feeder layers similar to embryonic stem cells ( Fig. 1A ). During proliferation, iPSC colonies expressed many typical human pluripotent markers (i.e. SOX2, TRA1-60 in Fig. 1A ), and expressed integrin (α3, α6 and β1 subunit) proteins, other NP markers (CD24 and Brachyury), as well as MSC markers (CD29, CD90). Once cultured under soft laminin-rich matrix conditions, iPSCs were shown to adopt a cell clustering morphology. At day 28 of differentiation, iPSCs expressed NP-related matrix proteins (laminin 10, LM511; type II collagen and proteoglycans as indicated by Saf O, Fig. 1B ), LM511 specific receptors (CD239, integrin subunit a3, a6 and b4, Fig. 1 B), as well as many NP markers (CD24, vimentin, cytokeratin 8 and N-cadherin, Fig. 1B ); it is noteworthy that type I collagen and E-cadherin were not expressed. [Figure: see text] Conclusion Our study evaluated a novel cell source that has potential use for cellular therapy in the IVD. Results demonstrate that native NP environment factors (soft laminin-rich matrix and secreted soluble factors) are able to promote human iPSC differentiation into a cell type exhibiting many NP-like markers and morphology. Future studies will focus on selection of NP-genic progenitor cells from these hiPSCs for IVD cellular therapy. Acknowledgments Supported by NIH R01AR057410, R01EB002263, R01AR047442,AOSpine Foundation and FAMRI Young Clinical Scientist award. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Nishimura, et al. Spine 1998;23:1531 Okuma, et al. Journal of Orthopaedic Research 2000;18:988 Meisel, et al. European Spine Journal 2006;15(S3):397 Sakai. Orthopaedic Clinics of North America. 2011;42:555 Yamanaka. Cell 2009;137:13 Chon, et al. Trans ORS 2011;36:600 Jing, et al. Tans ORS 2012;37: 31 Purmessur, et al. Arthritis Research Therapy 2011;13:R81

N-acetylcysteine protects induced pluripotent stem cells from in vitro stress: impact on differentiation outcome

Induced pluripotent stem cells (iPSCs) have the ability to differentiate towards various cell types of the adult organism and are a potential source of transplantable material in regenerative medicine. The entire process of conversion of iPSCs into terminally differentiated cells takes place in vitro and requires long periods of time. During in vitro culture, cells are exposed to environmental factors, which are capable of decreasing cellular performance and viability. Oxidative stress is the major underlying mechanism of such negative impact of in vitro environmental factors. We aimed to study the alteration of cellular properties during in vitro hematopoietic differentiation of human iPSCs and the ability of N-acetylcysteine (NAC), a potent free radical scavenger, to prevent such alterations. IPSCs were differentiated towards hematopoietic cells in the presence of 1 mM NAC. Intracellular reactive oxygen species (ROS), nitric oxide (NO), senescence, apoptosis and mitochondrial membrane potential (MMP) were evaluated at 1 and 3 weeks of differentiation. In the course of hematopoietic differentiation of iPSCs, cells progressively accumulated intracellular ROS and NO, increased the levels of apoptosis and senescence, and showed a decrease in mitochondrial functionality. NAC supplementation reversed all these phenomena. NAC administration also improved hematopoietic differentiation of iPSCs in terms of production of CD34, CD45 and CD43 positive cells. In conclusion, when supplemented during hematopoietic differentiation of iPSCs, NAC decreased oxidative stress, rescued the decline in cellular properties induced by long-term in vitro culture and promoted hematopoietic differentiation of iPSCs.

Abstract P092: Simplified Monolayer Differentiation of Human-Induced Pluripotent Stem Cells to Functional Cardiac Myocytes

Background: Human induced pluripotent stem cells (hiPSCs) are an important model for cardiovascular research, drug discovery, and translational research applications. Commonly used methods to direct iPSCs to cardiac myocytes can be technically demanding. Prior studies have shown that both VEGF and endothelial cells promote differentiation of stem cells to cardiac myocytes. Furthermore, DMEM/F12 with 10% fetal calf serum (DMEM-FCS) has been shown to induce cardiac myocytes in an embryoid body (EB) system. The objective of this study was to determine if differentiation of hiPSCs using conditions that support endothelial cell differentiation would promote cardiac myocyte colony formation. Methods: Two hiPSC lines derived using non-genome integrating methods were maintained on Matrigel-coated surfaces under serum free conditions in mTeSR1 medium. We performed a comparison of monolayer myocyte differentiation efficiency using DMEM-FCS and endothelial cell medium (EC). Cells were maintained in iPSC medium (mTeSR1) as a negative control. The number of beating colonies derived under each growth condition was determined using phase microscopy at 4 weeks. Cardiac myocyte commitment was characterized using an α-MHC-GFP reporter vector and electrophysiologic action potentials on isolated beating colonies. Results: Differentiation of human iPSCs in EC medium induced substantial numbers of beating colonies 4 weeks after differentiation (2.29 ± 0.3 beating colonies/cm2 culture area, n=42). Unlike EB models of myocyte differentiation, no beating clusters were observed in our monolayer system with DMEM-FCS medium (n=14) (p<0.01). As expected, mTESR1 (n=12) did not induce any cardiac myocytes. All beating cell colonies expressed GFP driven by the cardiac specific α-MHC promoter. Electrophysiological studies confirmed the presence of action potentials with ventricular phenotypes. Conclusions: Differentiation of human iPSCs under monolayer conditions that support endothelial cells facilitates efficient induction of functional human cardiac myocytes. Our findings simplify the differentiation of iPSCs to cardiac myocytes, making research with human iPSCs more accessible to a broad range of cardiovascular investigators.

Efficient Generation of Hepatoblasts From Human ES Cells and iPS Cells by Transient Overexpression of Homeobox Gene HEX

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed α-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.

Signaling Pathways Controlling Pluripotency and Early Cell Fate Decisions of Human Induced Pluripotent Stem Cells

Human pluripotent stem cells from embryonic origins and those generated from reprogrammed somatic cells share many characteristics, including indefinite proliferation and a sustained capacity to differentiate into a wide variety of cell types. However, it remains to be demonstrated whether both cell types rely on similar mechanisms to maintain their pluripotent status and to control their differentiation. Any differences in such mechanisms would suggest that reprogramming of fibroblasts to generate induced pluripotent stem cells (iPSCs) results in novel states of pluripotency. In that event, current methods for expanding and differentiating human embryonic stem cells (ESCs) might not be directly applicable to human iPSCs. However, we show here that human iPSCs rely on activin/nodal signaling to control Nanog expression and thereby maintain pluripotency, thus revealing their mechanistic similarity to human ESCs. We also show that growth factors necessary and sufficient for achieving specification of human ESCs into extraembryonic tissues, neuroectoderm, and mesendoderm also drive differentiation of human iPSCs into the same tissues. Importantly, these experiments were performed in fully chemically defined medium devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Together these data reveal that human iPSCs rely on mechanisms similar to human ESCs to maintain their pluripotency and to control their differentiation, showing that these pluripotent cell types are functionally equivalent.