Cell fate plug and play: direct reprogramming and induced pluripotency.

Building on the discovery that MyoD expression reprograms fibroblasts into muscle, three papers (Vierbuchen et al., 2010; Ieda et al., 2010; Szabo et al., 2010) recently reported the reprogramming of fibroblasts into neurons, cardiomyocytes, and blood cell progenitors without first passing the cells through a pluripotent state. Here we discuss the advantages and challenges of harnessing this direct reprogramming method for regenerative medicine.

TCF3 alternative splicing controlled by hnRNP H/F regulates E-cadherin expression and hESC pluripotency.

Alternative splicing (AS) plays important roles in embryonic stem cell (ESC) differentiation. In this study, we first identified transcripts that display specific AS patterns in pluripotent human ESCs (hESCs) relative to differentiated cells. One of these encodes T-cell factor 3 (TCF3), a transcription factor that plays important roles in ESC differentiation. AS creates two TCF3 isoforms, E12 and E47, and we identified two related splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNPs) H1 and F (hnRNP H/F), that regulate TCF3 splicing. We found that hnRNP H/F levels are high in hESCs, leading to high E12 expression, but decrease during differentiation, switching splicing to produce elevated E47 levels. Importantly, hnRNP H/F knockdown not only recapitulated the switch in TCF3 AS but also destabilized hESC colonies and induced differentiation. Providing an explanation for this, we show that expression of known TCF3 target E-cadherin, critical for maintaining ESC pluripotency, is repressed by E47 but not by E12.

Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.

The isolation of human induced pluripotent stem cells (iPSCs) offers a new strategy for modelling human disease. Recent studies have reported the derivation and differentiation of disease-specific human iPSCs. However, a key challenge in the field is the demonstration of disease-related phenotypes and the ability to model pathogenesis and treatment of disease in iPSCs. Familial dysautonomia (FD) is a rare but fatal peripheral neuropathy, caused by a point mutation in the IKBKAP gene involved in transcriptional elongation. The disease is characterized by the depletion of autonomic and sensory neurons. The specificity to the peripheral nervous system and the mechanism of neuron loss in FD are poorly understood owing to the lack of an appropriate model system. Here we report the derivation of patient-specific FD-iPSCs and the directed differentiation into cells of all three germ layers including peripheral neurons. Gene expression analysis in purified FD-iPSC-derived lineages demonstrates tissue-specific mis-splicing of IKBKAP in vitro. Patient-specific neural crest precursors express particularly low levels of normal IKBKAP transcript, suggesting a mechanism for disease specificity. FD pathogenesis is further characterized by transcriptome analysis and cell-based assays revealing marked defects in neurogenic differentiation and migration behaviour. Furthermore, we use FD-iPSCs for validating the potency of candidate drugs in reversing aberrant splicing and ameliorating neuronal differentiation and migration. Our study illustrates the promise of iPSC technology for gaining new insights into human disease pathogenesis and treatment.

Epigenetic changes and disturbed neural development in a human embryonic stem cell-based model relating to the fetal valproate syndrome.

Exposure to the antiepileptic drug valproic acid (VPA) during gestation causes neurofunctional and anatomic deficits in later life. At present, there are little human data on how early neural development is affected by chemicals. We used human embryonic stem cells, differentiating to neuroectodermal precursors, as a model to investigate the modes of action of VPA. Microarray expression profiling, qPCR of specific marker genes, immunostaining and the expression of green fluorescent protein under the control of the promoter of the canonical neural precursor cell marker HES5 were used as readouts. Exposure to VPA resulted in distorted marker gene expression, characterized by a relative increase in NANOG and OCT4 and a reduction in PAX6. A similar response pattern was observed with trichostatin A, a potent and specific histone deacetylase inhibitor (HDACi), but not with several other toxicants. Differentiation markers were disturbed by prolonged, but not by acute treatment with HDACi, and the strongest disturbance of differentiation was observed by toxicant exposure during early neural fate decision. The increased acetylation of histones observed in the presence of HDACi may explain the up-regulation of some genes. However, to understand the down-regulation of PAX6 and the overall complex transcript changes, we examined further epigenetic markers. Alterations in the methylation of lysines 4 and 27 of histone H3 were detected in the promoter region of PAX6 and OCT4. The changes in these activating and silencing histone marks provide a more general mechanistic rational for the regulation of developmentally important genes at non-cytotoxic drug concentrations.

Irgm1 protects hematopoietic stem cells by negative regulation of IFN signaling.

The IFN-inducible immunity-related p47 GTPase Irgm1 has been linked to Crohn disease as well as susceptibility to tuberculosis. Previously we demonstrated that HSC quiescence and function are aberrant in mice lacking Irgm1. To investigate the molecular basis for these defects, we conducted microarray expression profiling of Irgm1-deficient HSCs. Cell-cycle and IFN-response genes are up-regulated in Irgm1(-/-) HSCs, consistent with dysregulated IFN signaling. To test the hypothesis that Irgm1 normally down-regulates IFN signaling in HSCs, we generated Irgm1(-/-)Ifngr1(-/-) and Irgm1(-/-)Stat1(-/-) double-knockout animals. Strikingly, hyperproliferation, self-renewal, and autophagy defects in Irgm1(-/-) HSCs were normalized in double-knockout animals. These defects were also abolished in Irgm1(-/-)Irgm3(-/-) double-knockout animals, indicating that Irgm1 may regulate Irgm3 activity. Furthermore, the number of HSCs was reduced in aged Irgm1(-/-) animals, suggesting that negative feedback inhibition of IFN signaling by Irgm1 is necessary to prevent hyperproliferation and depletion of the stem cell compartment. Collectively, our results indicate that Irgm1 is a powerful negative regulator of IFN-dependent stimulation in HSCs, with an essential role in preserving HSC number and function. The deleterious effects of excessive IFN signaling may explain how hematologic abnormalities arise in patients with inflammatory conditions.

Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation.

Human-induced pluripotent stem cells (hiPSCs) are generated from somatic cells by ectopic expression of the 4 reprogramming factors (RFs) Oct-4, Sox2, Klf4, and c-Myc. To better define the stoichiometric requirements and dynamic expression patterns required for successful hiPSC induction, we generated 4 bicistronic lentiviral vectors encoding the 4 RFs co-expressed with discernable fluorescent proteins. Using this system, we define the optimal stoichiometry of RF expression to be highly sensitive to Oct4 dosage, and we demonstrate the impact that variations in the relative ratios of RF expression exert on the efficiency of hiPSC induction. Monitoring of expression of each individual RF in single cells during the course of reprogramming revealed that vector silencing follows acquisition of pluripotent cell markers. Pronounced lentiviral vector silencing was a characteristic of successfully reprogrammed hiPSC clones, but lack of complete silencing did not hinder hiPSC induction, maintenance, or directed differentiation. The vector system described here presents a powerful tool for mechanistic studies of reprogramming and the optimization of hiPSC generation.

Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation.

Age-related defects in stem cells can limit proper tissue maintenance and hence contribute to a shortened lifespan. Using highly purified hematopoietic stem cells from mice aged 2 to 21 mo, we demonstrate a deficit in function yet an increase in stem cell number with advancing age. Expression analysis of more than 14,000 genes identified 1,500 that were age-induced and 1,600 that were age-repressed. Genes associated with the stress response, inflammation, and protein aggregation dominated the up-regulated expression profile, while the down-regulated profile was marked by genes involved in the preservation of genomic integrity and chromatin remodeling. Many chromosomal regions showed coordinate loss of transcriptional regulation; an overall increase in transcriptional activity with age and inappropriate expression of genes normally regulated by epigenetic mechanisms was also observed. Hematopoietic stem cells from early-aging mice expressing a mutant p53 allele reveal that aging of stem cells can be uncoupled from aging at an organismal level. These studies show that hematopoietic stem cells are not protected from aging. Instead, loss of epigenetic regulation at the chromatin level may drive both functional attenuation of cells, as well as other manifestations of aging, including the increased propensity for neoplastic transformation.

Lipid Deprivation Induces a Stable, Naive-to-Primed Intermediate State of Pluripotency in Human PSCs.

Current challenges in capturing naive human pluripotent stem cells (hPSCs) suggest that the factors regulating human naive versus primed pluripotency remain incompletely defined. Here we demonstrate that the widely used Essential 8 minimal medium (E8) captures hPSCs at a naive-to-primed intermediate state of pluripotency expressing several naive-like developmental, bioenergetic, and epigenomic features despite providing primed-state-sustaining growth factor conditions. Transcriptionally, E8 hPSCs are marked by activated lipid biosynthesis and suppressed MAPK/TGF-ß gene expression, resulting in endogenous ERK inhibition. These features are dependent on lipid-free culture conditions and are lost upon lipid exposure, whereas short-term pharmacological ERK inhibition restores naive-to-primed intermediate traits even in the presence of lipids. Finally, we identify de novo lipogenesis as a common transcriptional signature of E8 hPSCs and the pre-implantation human epiblast in vivo. These findings implicate exogenous lipid availability in regulating human pluripotency and define E8 hPSCs as a stable, naive-to-primed intermediate (NPI) pluripotent state.

Dual-SMAD Inhibition/WNT Activation-Based Methods to Induce Neural Crest and Derivatives from Human Pluripotent Stem Cells.

The neural crest (NC) is a transient population of multipotent cells giving rise to the peripheral nervous system, skin pigmentation, heart, and facial mesenchyme. The broad cell fate potential of NC makes it an attractive cell fate to derive from human pluripotent stem cells (hPSCs) for exploring embryonic development, modeling disease, and generating cells for transplantation. Here, we discuss recent publications and methods for efficiently differentiating hPSCs into NC. We also provide methods to direct NC into two different terminal fates: melanocytes and sensory neurons.

Build-a-brain.

A major barrier in understanding nervous system development is modeling the cellular interactions that form the human brain. Recently, in the journal Nature, Lancaster et al. (2013) established a protocol for culturing pluripotent stem cell (PSC)-derived "cerebral organoids" that mimics the developing human brain's cellular organization, segregates into distinct brain regions, and models microcephaly.

Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs.

Melanocytes are pigment-producing cells of neural crest (NC) origin that are responsible for protecting the skin against UV irradiation. Pluripotent stem cell (PSC) technology offers a promising approach for studying human melanocyte development and disease. Here, we report that timed exposure to activators of WNT, BMP, and EDN3 signaling triggers the sequential induction of NC and melanocyte precursor fates under dual-SMAD-inhibition conditions. Using a SOX10::GFP human embryonic stem cell (hESC) reporter line, we demonstrate that the temporal onset of WNT activation is particularly critical for human NC induction. Subsequent maturation of hESC-derived melanocytes yields pure populations that match the molecular and functional properties of adult melanocytes. Melanocytes from Hermansky-Pudlak syndrome and Chediak-Higashi syndrome patient-specific induced PSCs (iPSCs) faithfully reproduce the ultrastructural features of disease-associated pigmentation defects. Our data define a highly specific requirement for WNT signaling during NC induction and enable the generation of pure populations of human iPSC-derived melanocytes for faithful modeling of pigmentation disorders.

ZFX controls the self-renewal of human embryonic stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer great promise in regenerative medicine and disease modeling due to their unlimited self-renewal and broad differentiation capacity. There is evidence that the growth properties and critical signaling pathways differ between murine and human ESCs; therefore, it is essential to perform functional studies to test the putatively conserved mechanisms of pluripotent stem cell self-renewal between species. Previously, we identified the transcription factor Zfx as a key regulator of self-renewal in murine ESCs. Here we extend those findings to human ESCs. ZFX knockdown in hESCs hindered clonal growth and decreased colony size after serial replating. ZFX overexpression enhanced clone formation in the presence of Y-27632, increased colony size at low density and decreased expression of differentiation-related genes in human ESCs. ZFX-overexpressing hESCs resisted spontaneous differentiation but could be directed to differentiate into endodermal and neural cell fates when provided with the appropriate cues. Thus, ZFX acts as a molecular rheostat regulating the balance between self-renewal and differentiation in hESCs, revealing the close evolutionary conservation of the self-renewal mechanisms in murine and human ESCs.

Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors.

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 >75% 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.

Converting human pluripotent stem cells to neural tissue and neurons to model neurodegeneration.

Human embryonic stem cells (hESCs) and the related induced pluripotent stem cells (hiPSCs) have attracted considerable attention since they can provide an unlimited source of many different tissue types. One challenge of using pluripotent cells is directing their broad differentiation potential into one specific tissue or cell fate. The cell fate choices of extraembryonic, endoderm, mesoderm, and ectoderm (including neural) lineages represent the earliest decisions. We found that pluripotent cells efficiently neuralize by blocking the signaling pathways required for alternative cell fate decisions. In this chapter, we detail methods to direct hESCs or hiPSCs into early neural cells and subsequently postmitotic neurons.

Derivation of neural crest cells from human pluripotent stem cells.

Human pluripotent stem cell (hPSC)-derived neural crest (NC) cells present a valuable tool for modeling aspects of human NC development, including cell fate specification, multipotency and cell migration. hPSC-derived NC cells are also suitable for modeling human disease and as a renewable cell source for applications in regenerative medicine. Here we provide protocols for the step-wise differentiation of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into neuroectodermal and NC cells using either the MS5 coculture system or a novel defined culture method based on pharmacological inhibition of bone morphogenetic protein and transforming growth factor-beta signaling pathways. Furthermore, we present protocols for the purification and propagation of hPSC-NC cells using flow cytometry and defined in vitro culture conditions. Our protocol has been validated in multiple independent hESC and hiPSC lines. The average time required for generating purified hPSC-NC precursors using this protocol is 2-5 weeks.

Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-beta1.

The traditional view of hematopoiesis has been that all the cells of the peripheral blood are the progeny of a unitary homogeneous pool of hematopoietic stem cells (HSCs). Recent evidence suggests that the hematopoietic system is actually maintained by a consortium of HSC subtypes with distinct functional characteristics. We show here that myeloid-biased HSCs (My-HSCs) and lymphoid-biased HSCs (Ly-HSCs) can be purified according to their capacity for Hoechst dye efflux in combination with canonical HSC markers. These phenotypes are stable under natural (aging) or artificial (serial transplantation) stress and are exacerbated in the presence of competing HSCs. My- and Ly-HSCs respond differently to TGF-beta1, presenting a possible mechanism for differential regulation of HSC subtype activation. This study demonstrates definitive isolation of lineage-biased HSC subtypes and contributes to the fundamental change in view that the hematopoietic system is maintained by a continuum of HSC subtypes, rather than a functionally uniform pool.

Efficient derivation of functional floor plate tissue from human embryonic stem cells.

The floor plate (FP) is a critical signaling center during neural development located along the ventral midline of the embryo. Little is known about human FP development because of the lack of tissue accessibility. Here we report the efficient derivation of human embryonic stem cell (hESC)-derived FP tissue capable of secreting Netrin-1 and SHH and patterning primary and hESC derived tissues. FP induction in hESCs is dependent on early SHH exposure and occurs at the expense of anterior neurectoderm (AN). Global gene expression and functional studies identify SHH-mediated inhibition of Dkk-1 as key factor in FP versus AN specification. hESC-derived FP tissue is shown to be of anterior SIX6+ character but is responsive to caudalizing factors suppressing SIX6 expression and inducing a shift in usage of region-specific SHH enhancers. These data define the early signals that drive human FP versus AN specification and determine regional identity in hESC-derived FP.

Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.

Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient to induce rapid and complete neural conversion of >80% of hES cells under adherent culture conditions. Temporal fate analysis reveals the appearance of a transient FGF5(+) epiblast-like stage followed by PAX6(+) neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.