Scientific Resources

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions,the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4,Nanog,Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl(2) (n = 22). Patch-clamp techniques were used to record I(Kr) and I(K1) from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (ptextless0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC,E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl(2),at concentrations that selectively block I(K1) (50-100 µM),failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I(K1) in 53% (20/38) of the cells studied,but presence of I(Kr) in all (11/11). Consistent with the electrophysiological data,RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I(K1) in the majority of cells,but robust expression of I(Kr.) In contrast to recently reported studies,our data point to major deficiencies of hiPSC-CM,with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I(Kr) due to the absence of I(K1). Thus,efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications. View Publication

We developed a protocol of in vitro differentiation of human embryonic stem cells into three-dimensional structures histologically and molecularly similar to the developing retina. View Publication

Stable pluripotent feeder-free propagation of human embryonic stem cells (hESCs) prior to their therapeutic applications remains a major challenge. Matrigel™ (BD Singapore) is a murine sarcoma-derived extracellular matrix (ECM) widely used as a cell-free support combined with conditioned or chemically defined media; however,inherent xenogenic and immunological threats invalidate it for clinical applications. Using human fibrogenic cells to generate ECM is promising but currently suffers from inefficient and time-consuming deposition in vitro. We recently showed that macromolecular crowding (MMC) accelerated ECM deposition substantially in vitro. In the current study,we used dextran sulfate 500 kDa as a macromolecular crowder to induce WI-38 fetal human lung fibroblasts at 0.5% serum condition to deposit human ECM in three days. After decellularization,the generated ECMs allowed stable propagation of H9 hESCs over 20 passages in chemically-defined medium (mTEsR1) with an overall improved outcome compared to Matrigel in terms of population doubling while retaining teratoma formation and differentiation capacity. Of significance,only ECMs generated by MMC allowed the successful propagation of hESCs. ECMs were highly complex and in contrast to Matrigel,contained no vitronectin but did contain collagen XII,ig-h3 and novel for hESC-supporting human matrices,substantial amounts of transglutaminase 2. Genome-wide analysis of promoter DNA methylation states revealed high overall similarity between human ECM- and Matrigel-cultured hESCs; however,distinct differences were observed with 49 genes associated with a variety of cellular functions. Thus,human ECMs deposited by MMC by selected fibroblast lines are a suitable human microenvironment for stable hESC propagation and clinically translational settings. View Publication

Based on knowledge of early embryo development,where anterior neural ectoderm (ANE) development is regulated by native inhibitors of bone morphogenic protein (BMP) and Nodal/Activin signaling,most published protocols of human embryonic stem cell differentiation to ANE have demonstrated a crucial role for Smad signaling in neural induction. The drawbacks of such protocols include the use of an embryoid body culture step and use of polypeptide secreted factors that are both expensive and,when considering clinical applications,have significant challenges in terms of good manufacturing practices compliancy. The use of small molecules to direct differentiation of pluripotent stem cells toward a specified lineage represents a powerful approach to generate specific cell types for further understanding of biological function,for understanding disease processes,for use in drug discovery,and finally for use in regenerative medicine. We therefore aimed to find controlled and reproducible animal-component-free differentiation conditions that would use only small molecules. Here,we demonstrate that pluripotent stem cells can be reproducibly and efficiently differentiated to PAX6(+) (a marker of neuroectoderm) and OCT4(-) (a marker of pluripotent stem cells) cells with the use of potent small inhibitors of the BMP and Activin/Nodal pathways,and in animal-component-free conditions,replacing the frequently used Noggin and SB431542. We also show by transcript analysis,both at the population level and for the first time at the single-cell level,that differentiated cells express genes characteristic for the development of ANE,in particular for the development of the future forebrain. View Publication

Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However,due to risks of random integration of the reprogramming transgenes into the host genome,the low efficiency of the process,and the potential risk of virally induced tumorigenicity,alternative methods have been developed to generate pluripotent cells using nonintegrating systems,albeit with limited success. Here,we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors,by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion,they maintain genetic stability,protein level expression of key pluripotency factors,high cell-division kinetics,telomerase activity,repression of X-inactivation,and capacity to differentiate into lineages of the three germ layers,such as definitive endoderm,hepatocytes,bone,fat,cartilage,neurons,and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies,pharmaceutical screening,and disease modeling. View Publication

Considerable progress has been made in identifying signaling pathways that direct the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types,including neurons. However,differentiation of hPSCs with extrinsic factors is a slow,step-wise process,mimicking the protracted timing of human development. Using a small-molecule screen,we identified a combination of five small-molecule pathway inhibitors that yield hPSC-derived neurons at textgreater75% efficiency within 10 d of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors,including tetrodotoxin (TTX)-resistant,SCN10A-dependent sodium currents and response to nociceptive stimuli such as ATP and capsaicin. Neuronal fate acquisition occurs about threefold faster than during in vivo development,suggesting that use of small-molecule pathway inhibitors could become a general strategy for accelerating developmental timing in vitro. The quick and high-efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain. View Publication

Long QT syndrome (LQTS) is a genetic disease characterized by a prolonged QT interval in an electrocardiogram (ECG),leading to higher risk of sudden cardiac death. Among the 12 identified genes causal to heritable LQTS,∼90% of affected individuals harbor mutations in either KCNQ1 or human ether-a-go-go related genes (hERG),which encode two repolarizing potassium currents known as I(Ks) and I(Kr). The ability to quantitatively assess contributions of different current components is therefore important for investigating disease phenotypes and testing effectiveness of pharmacological modulation. Here we report a quantitative analysis by simulating cardiac action potentials of cultured human cardiomyocytes to match the experimental waveforms of both healthy control and LQT syndrome type 1 (LQT1) action potentials. The quantitative evaluation suggests that elevation of I(Kr) by reducing voltage sensitivity of inactivation,not via slowing of deactivation,could more effectively restore normal QT duration if I(Ks) is reduced. Using a unique specific chemical activator for I(Kr) that has a primary effect of causing a right shift of V(1/2) for inactivation,we then examined the duration changes of autonomous action potentials from differentiated human cardiomyocytes. Indeed,this activator causes dose-dependent shortening of the action potential durations and is able to normalize action potentials of cells of patients with LQT1. In contrast,an I(Kr) chemical activator of primary effects in slowing channel deactivation was not effective in modulating action potential durations. Our studies provide both the theoretical basis and experimental support for compensatory normalization of action potential duration by a pharmacological agent. View Publication

Human embryonic stem cells (hESCs) hold great promise for regenerative medicine because they can undergo unlimited self-renewal and retain the capability to differentiate into all cell types in the body. Although numerous genes/proteins such as Oct4 and Gata6 have been identified to play critical regulatory roles in self-renewal and differentiation of hESC,the majority of the regulators in these cellular processes and more importantly how these regulators co-operate with each other and/or with epigenetic modifications are still largely unknown. We propose here a systematic approach to integrate genomic and epigenomic data for identification of direct regulatory interactions. This approach allows reconstruction of cell-type-specific transcription networks in embryonic stem cells (ESCs) and fibroblasts at an unprecedented scale. Many links in the reconstructed networks coincide with known regulatory interactions or literature evidence. Systems-level analyses of these networks not only uncover novel regulators for pluripotency and differentiation,but also reveal extensive interplays between transcription factor binding and epigenetic modifications. Especially,we observed poised enhancers characterized by both active (H3K4me1) and repressive (H3K27me3) histone marks that contain enriched Oct4- and Suz12-binding sites. The success of such a systems biology approach is further supported by experimental validation of the predicted interactions. View Publication

The blood-brain barrier (BBB) is crucial to the health of the brain and is often compromised in neurological disease. Moreover,because of its barrier properties,this endothelial interface restricts uptake of neurotherapeutics. Thus,a renewable source of human BBB endothelium could spur brain research and pharmaceutical development. Here we show that endothelial cells derived from human pluripotent stem cells (hPSCs) acquire BBB properties when co-differentiated with neural cells that provide relevant cues,including those involved in Wnt/β-catenin signaling. The resulting endothelial cells have many BBB attributes,including well-organized tight junctions,appropriate expression of nutrient transporters and polarized efflux transporter activity. Notably,they respond to astrocytes,acquiring substantial barrier properties as measured by transendothelial electrical resistance (1,450 ± 140 Ω cm2),and they possess molecular permeability that correlates well with in vivo rodent blood-brain transfer coefficients. View Publication

The discovery of induced pluripotent stem cells(iPSCs) is a promising advancement in the field of regenerative medicine. Previous studies have indicated that the teratoma-forming propensity of iPSCs is variable; however,the relationship between tumorigenic potential and genomic instability in human iPSCs (HiPSCs) remains to be fully elucidated. Here,we evaluated the malignant potential of HiPSCs by using both colony formation assays and tumorigenicity tests. We demonstrated that HiPSCs formed tumorigenic colonies when grown in cancer cell culture medium and produced malignancies in immunodeficient mice. Furthermore,we analyzed genomic instability in HiPSCs using whole-genome copy number variation analysis and determined that the extent of genomic instability was related with both the cells' propensity to form colonies and their potential for tumorigenesis. These findings indicate a risk for potential malignancy of HiPSCs derived from genomic instability and suggest that quality control tests,including comprehensive tumorigenicity assays and genomic integrity validation,should be rigorously executed before the clinical application of HiPSCs. In addition,HiPSCs should be generated through the use of combined factors or other approaches that decrease the likelihood of genomic instability. View Publication

Cellular reprogramming from adult somatic cells into an embryonic cell-like state,termed induced pluripotency,has been achieved in several cell types. However,the ability to reprogram human amniotic epithelial cells (hAECs),an abundant cell source derived from discarded placental tissue,has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs),but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore,AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation,including NEUROD1 and SOX17,markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs,we analyzed global DNA methylation,global histone acetylation,and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts,hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise,quantitative gene expression analyses show that hAECs endogenously express OCT4,SOX2,KLF4,and c-MYC,all four factors used in cellular reprogramming. Thus,hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents. View Publication

PURPOSE:
Human embryonic stem cells (hESC) hold a great potential for regenerative medicine because,in principle,they can differentiate into any cell type found in the human body. In addition,studying the effect of ionizing radiation (IR) on hESC may provide valuable information about the response of human cells to IR exposure in their most naive state,as well as the consequences of IR exposure on the development of organisms. However,the effect of IR,in particular radionuclide uptake,on the pluripotency,proliferation and survival of hESC has not been extensively studied.
METHODS:
In this study we treated cultured hESC with 5-[(125)I]iodo-2'-deoxyuridine ((125)IdU),a precursor of DNA synthesis. Then we measured the expansion of colonies and expression of pluripotency markers in hESC.
RESULTS:
We found that uptake of (125)IdU was similar in both hESC and HT1080 human fibrosarcoma cells. However,treatment with 0.1 μCi/ml (125)IdU for 24 hours resulted in complete death of the hESC population; whereas HT1080 cancer cells continued to grow. Treatment with a 10-fold lower dose (125)IdU (0.01 μCi/ml) resulted in colonies of hESC becoming less defined with numerous cells growing in monolayer outside of the colonies showing signs of differentiation. Then we analyzed the expression of pluripotency markers (octamer-binding transcription factor 4 [Oct-4] and stage-specific embryonic antigen-4 [SSEA4]) in the surviving hESC. We found that hESC in the surviving colonies expressed pluripotency markers at levels comparable with those in the non-treated controls.
CONCLUSIONS:
Our results provide important initial insights into the sensitivity of hESC to IR,and especially that produced by the decay of an internalized radionuclide. View Publication