Coating-Free Culture Medium for Establishing and Maintaining Human Induced Pluripotent Stem Cells.

Cell expansion of human pluripotent stem cells (hPSCs) commonly depends on Matrigel as a coating matrix on two-dimensional (2D) culture plates and 3D microcarriers. However, the xenogenic Matrigel requires sophisticated quality-assurance processes to meet clinical requirements. In this study, we develop an innovative coating-free medium for expanding hPSCs. The xenofree medium supports the weekend-free culture and competitive growth of hPSCs on several cell culture plastics without an additional pre-coating process. The pluripotent stemness of the expanded cells is stably sustained for more than 10 passages, featured with high pluripotent marker expressions, normal karyotyping, and differentiating capacity for three germ layers. The expression levels of some integrins are reduced, compared with those of the hPSCs on Matrigel. This medium also successfully supports the clonal expansion and induced pluripotent stem cell establishment from mitochondrial-defective MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) patient's peripheral blood mononuclear cells. This innovative hPSC medium provides a straightforward scale-up process for producing clinical-orientated hPSCs by excluding the conventional coating procedure.

Cardiac myofibrillogenesis is spatiotemporally modulated by the molecular chaperone UNC45B.

Sarcomeres are fundamental to cardiac muscle contraction. Their impairment can elicit cardiomyopathies, leading causes of death worldwide. However, the molecular mechanism underlying sarcomere assembly remains obscure. We used human embryonic stem cell (hESC)-derived cardiomyocytes (CMs) to reveal stepwise spatiotemporal regulation of core cardiac myofibrillogenesis-associated proteins. We found that the molecular chaperone UNC45B is highly co-expressed with KINDLIN2 (KIND2), a marker of protocostameres, and later its distribution overlaps with that of muscle myosin MYH6. UNC45B-knockout CMs display essentially no contractility. Our phenotypic analyses further reveal that (1) binding of Z line anchor protein ACTN2 to protocostameres is perturbed because of impaired protocostamere formation, resulting in ACTN2 accumulation; (2) F-ACTIN polymerization is suppressed; and (3) MYH6 becomes degraded, so it cannot replace non-muscle myosin MYH10. Our mechanistic study demonstrates that UNC45B mediates protocostamere formation by regulating KIND2 expression. Thus, we show that UNC45B modulates cardiac myofibrillogenesis by interacting spatiotemporally with various proteins.

UQCRC1 engages cytochrome c for neuronal apoptotic cell death.

Human ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) is an evolutionarily conserved core subunit of mitochondrial respiratory chain complex III. We recently identified the disease-associated variants of UQCRC1 from patients with familial parkinsonism, but its function remains unclear. Here we investigate the endogenous function of UQCRC1 in the human neuronal cell line and the Drosophila nervous system. Flies with neuronal knockdown of uqcrc1 exhibit age-dependent parkinsonism-resembling defects, including dopaminergic neuron reduction and locomotor decline, and are ameliorated by UQCRC1 expression. Lethality of uqcrc1-KO is also rescued by neuronally expressing UQCRC1, but not the disease-causing variant, providing a platform to discern the pathogenicity of this mutation. Furthermore, UQCRC1 associates with the apoptosis trigger cytochrome c (cyt-c), and uqcrc1 deficiency increases cyt-c in the cytoplasmic fraction and activates the caspase cascade. Depleting cyt-c or expression of the anti-apoptotic p35 ameliorates uqcrc1-mediated neurodegeneration. Our findings identify a role for UQCRC1 in regulating cyt-c-induced apoptosis.

Hypochlorite-induced porcine model of peritoneal fibrosis through the activation of IL1ß-CX3CL1-TGFß1 signal axis.

Patients with kidney failure rely on life-saving peritoneal dialysis to facilitate waste exchange and maintain homeostasis of physical conditions. However, peritoneal dialysis often results in peritoneal fibrosis and organ adhesion that subsequently compromise the efficiency of peritoneal dialysis and normal functions of visceral organs. Despite rodent models provide clues on the pathogenesis of peritoneal fibrosis, no current large animal model which shares high degree of physiological and anatomical similarities to human is available, limiting their applications on the evaluation of pre-clinical therapeutic efficacy. Here we established for the first time, hypochlorite-induced porcine model of peritoneal fibrosis in 5-week-old piglets. We showed that administration 15-30 mM hypochlorite, a dose- and time-dependent severity of peritoneal fibrosis characterized by mesothelium fragmentation, αSMA+ myofibroblasts accumulation, organ surface thickening and type I collagen deposition were observed. We also demonstrated in vitro using human mesothelial cells that hypochlorite-induced fibrosis was likely due to necrosis, but not programmed apoptosis; besides, overexpression of IL1ß, CX3CL1 and TGFß on the peritoneal mesothelium in current model was detected, similar to observations from peritoneal dialysis-induced peritoneal fibrosis in human patients and earlier reported mouse model. Moreover, our novel antemortem evaluation using laparoscopy provided instant feedback on the progression of organ fibrosis/adhesion which allows immediate adjustments on treatment protocols and strategies in alive individuals that can not and never be performed in other animal models.

Corrigendum: Colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing.

This corrects the article DOI: 10.1038/nm.4355.

Colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing.

With the goal of modeling human disease of the large intestine, we sought to develop an effective protocol for deriving colonic organoids (COs) from differentiated human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). Extensive gene and immunohistochemical profiling confirmed that the derived COs represent colon rather than small intestine, containing stem cells, transit-amplifying cells, and the expected spectrum of differentiated cells, including goblet and endocrine cells. We applied this strategy to iPSCs derived from patients with familial adenomatous polyposis (FAP-iPSCs) harboring germline mutations in the WNT-signaling-pathway-regulator gene encoding APC, and we generated COs that exhibit enhanced WNT activity and increased epithelial cell proliferation, which we used as a platform for drug testing. Two potential compounds, XAV939 and rapamycin, decreased proliferation in FAP-COs, but also affected cell proliferation in wild-type COs, which thus limits their therapeutic application. By contrast, we found that geneticin, a ribosome-binding antibiotic with translational 'read-through' activity, efficiently targeted abnormal WNT activity and restored normal proliferation specifically in APC-mutant FAP-COs. These studies provide an efficient strategy for deriving human COs, which can be used in disease modeling and drug discovery for colorectal disease.

Efficient Generation of Cardiac Purkinje-like Cells from Embryonic Stem Cells by Activating cAMP Signaling.

Strategies to derive cardiac conduction system (CCS) cells including Purkinje cells (PC) would facilitate models for mechanistic studies and drug discovery, and also provide new cellular materials for regenerative therapies. However, using current cardiac differentiation protocols, the differentiation efficiency of CCS cells is extremely low, typically below 1% of the culture. High-throughput chemical screening is a powerful strategy for identifying small molecules that can activate signaling pathways to enhance embryonic stem cell (ESC) differentiation. We describe how to carry out a high- throughput screen to identify small molecules that can efficiently promote CCS generation from mouse ESCs. We also describe several assays, including immunofluorescence staining, electrophysiology, FACS analysis and quantitative real- time PCR, to characterize the phenotype of ESC-derived PC. In summary, we describe an efficient way to identify small molecules that enhance cardiac PC generation. These protocols can be adapted to identify other rare cell lineages by directed differentiation from ESCs. © 2017 by John Wiley & Sons, Inc.

Modeling Cystic Fibrosis Using Pluripotent Stem Cell-Derived Human Pancreatic Ductal Epithelial Cells.

UNLABELLED: We established an efficient strategy to direct human pluripotent stem cells, including human embryonic stem cells (hESCs) and an induced pluripotent stem cell (iPSC) line derived from patients with cystic fibrosis, to differentiate into pancreatic ductal epithelial cells (PDECs). After purification, more than 98% of hESC-derived PDECs expressed functional cystic fibrosis transmembrane conductance regulator (CFTR) protein. In addition, iPSC lines were derived from a patient with CF carrying compound frameshift and mRNA splicing mutations and were differentiated to PDECs. PDECs derived from Weill Cornell cystic fibrosis (WCCF)-iPSCs showed defective expression of mature CFTR protein and impaired chloride ion channel activity, recapitulating functional defects of patients with CF at the cellular level. These studies provide a new methodology to derive pure PDECs expressing CFTR and establish a "disease in a dish" platform to identify drug candidates to rescue the pancreatic defects of patients with CF. SIGNIFICANCE: An efficient strategy was established to direct human pluripotent stem cells, including human embryonic stem cells (hESCs) and an induced pluripotent stem cell line derived from patients with cystic fibrosis (CF-iPSCs), to differentiate into pancreatic ductal epithelial cells (PDECs). After purification, more than 98% of hESC-PDECs derived from CF-iPSCs showed defective expression of mature cystic fibrosis transmembrane conductance regulator (CFTR) protein and impaired chloride ion channel activity, recapitulating functional pancreatic defects of patients with CF at the cellular level. These studies provide a new methodology for deriving pure PDECs expressing CFTR, and they establish a "disease-in-a-dish" platform for identifying drug candidates to rescue the pancreatic defects of these patients.

Efficient Generation of Cardiac Purkinje Cells from ESCs by Activating cAMP Signaling.

Dysfunction of the specialized cardiac conduction system (CCS) is associated with life-threatening arrhythmias. Strategies to derive CCS cells, including rare Purkinje cells (PCs), would facilitate models for mechanistic studies and drug discovery and also provide new cellular materials for regenerative therapies. A high-throughput chemical screen using CCS:lacz and Contactin2:egfp (Cntn2:egfp) reporter embryonic stem cell (ESC) lines was used to discover a small molecule, sodium nitroprusside (SN), that efficiently promotes the generation of cardiac cells that express gene profiles and generate action potentials of PC-like cells. Imaging and mechanistic studies suggest that SN promotes the generation of PCs from cardiac progenitors initially expressing cardiac myosin heavy chain and that it does so by activating cyclic AMP signaling. These findings provide a strategy to derive scalable PCs, along with insight into the ontogeny of CCS development.

Tbx18 targets dermal condensates for labeling, isolation, and gene ablation during embryonic hair follicle formation.

How cell fate decisions of stem and progenitor cells are regulated by their microenvironment or niche is a central question in stem cell and regenerative biology. Although functional analysis of hair follicle epithelial stem cells by gene targeting is well established, the molecular and genetic characterization of the dermal counterpart during embryonic morphogenesis has been lacking because of the absence of cell type-specific drivers. Here, we report that T-box transcription factor Tbx18 specifically marks dermal papilla (DP) precursor cells during embryonic hair follicle morphogenesis. With Tbx18(LacZ), Tbx18(H2BGFP), and Tbx18(Cre) knock-in mouse models, we demonstrate LacZ and H2BGFP (nuclear green fluorescent protein) expression and Cre activity in dermal condensates of nascent first-wave hair follicles at E14.5. As Tbx18 expression becomes more widespread throughout the dermis at later developmental stages, we use tamoxifen-inducible Cre-expressing mice, Tbx18(MerCreMer), to exclusively target DP precursor cells and their progeny. Finally, we ablate Tbx18 in full knockout mice, but find no perturbations in hair follicle formation, suggesting that Tbx18 is dispensable for normal DP function. In summary, our study establishes Tbx18 as a genetic driver to target for the first time embryonic DP precursors for labeling, isolation, and gene ablation that will greatly enhance investigations into their molecular functions during hair follicle morphogenesis.

Single transcription factor reprogramming of hair follicle dermal papilla cells to induced pluripotent stem cells.

Reprogramming patient-specific somatic cells into induced pluripotent stem (iPS) cells has great potential to develop feasible regenerative therapies. However, several issues need to be resolved such as ease, efficiency, and safety of generation of iPS cells. Many different cell types have been reprogrammed, most conveniently even peripheral blood mononuclear cells. However, they typically require the enforced expression of several transcription factors, posing mutagenesis risks as exogenous genetic material. To reduce this risk, iPS cells were previously generated with Oct4 alone from rather inaccessible neural stem cells that endogenously express the remaining reprogramming factors and very recently from fibroblasts with Oct4 alone in combination with additional small molecules. Here, we exploit that dermal papilla (DP) cells from hair follicles in the skin express all but one reprogramming factors to show that these accessible cells can be reprogrammed into iPS cells with the single transcription factor Oct4 and without further manipulation. Reprogramming was already achieved after 3 weeks and with efficiencies similar to other cell types reprogrammed with four factors. Dermal papilla-derived iPS cells are comparable to embryonic stem cells with respect to morphology, gene expression, and pluripotency. We conclude that DP cells may represent a preferred cell type for reprogramming accessible cells with less manipulation and for ultimately establishing safe conditions in the future by replacing Oct4 with small molecules.

Wdr5 mediates self-renewal and reprogramming via the embryonic stem cell core transcriptional network.

The embryonic stem (ES) cell transcriptional and chromatin-modifying networks are critical for self-renewal maintenance. However, it remains unclear whether these networks functionally interact and, if so, what factors mediate such interactions. Here, we show that WD repeat domain 5 (Wdr5), a core member of the mammalian Trithorax (trxG) complex, positively correlates with the undifferentiated state and is a regulator of ES cell self-renewal. We demonstrate that Wdr5, an "effector" of H3K4 methylation, interacts with the pluripotency transcription factor Oct4. Genome-wide protein localization and transcriptome analyses demonstrate overlapping gene regulatory functions between Oct4 and Wdr5. The Oct4-Sox2-Nanog circuitry and trxG cooperate in activating transcription of key self-renewal regulators, and furthermore, Wdr5 expression is required for the efficient formation of induced pluripotent stem (iPS) cells. We propose an integrated model of transcriptional and epigenetic control, mediated by select trxG members, for the maintenance of ES cell self-renewal and somatic cell reprogramming.