Scientific Resources

Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4,KLF4,SOX2,LIN28,cMYC and nuclear GFP over 18 days,then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4,NANOG,TRA-1-60,and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal,ectodermal,and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation,cells contracted spontaneously,expressed connexin 43 and β-myosin heavy chain organized in sarcomeric banding patterns,expressed cardiac troponin T and β-myosin heavy chain,showed upregulation of NKX2.5,ISL-1 and cardiac troponin T with downregulation of POU5F1,and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes. View Publication

The ability to create 3D tissues from induced pluripotent stem cells (iPSCs) is poised to revolutionize stem cell research and regenerative medicine,including individualized,patient-specific stem cell-based treatments. There are,however,few examples of tissue engineering using iPSCs. Their culture and differentiation is predominantly planar for monolayer cell support or induction of self-organizing embryoids (EBs) and organoids. Bioprinting iPSCs with advanced biomaterials promises to augment efforts to develop 3D tissues,ideally comprising direct-write printing of cells for encapsulation,proliferation,and differentiation. Here,such a method,employing a clinically amenable polysaccharide-based bioink,is described as the first example of bioprinting human iPSCs for in situ expansion and sequential differentiation. Specifically,There are extrusion printed the bioink including iPSCs,alginate (Al; 5% weight/volume [w/v]),carboxymethyl-chitosan (5% w/v),and agarose (Ag; 1.5% w/v),crosslinked the bioink in calcium chloride for a stable and porous construct,proliferated the iPSCs within the construct and differentiated the same iPSCs into either EBs comprising cells of three germ lineages-endoderm,ectoderm,and mesoderm,or more homogeneous neural tissues containing functional migrating neurons and neuroglia. This defined,scalable,and versatile platform is envisaged being useful in iPSC research and translation for pharmaceuticals development and regenerative medicine. View Publication

Genome-wide association studies (GWASs) have identified many disease-associated variant alleles,but understanding whether and how different genes/loci interact requires a platform for probing how the variant alleles act mechanistically. Isogenic mutant human embryonic stem cells (hESCs) provide an unlimited resource to derive and study human disease-relevant cells. Here,we focused on CDKAL1,linked by GWASs to diabetes. Through transcript profiling,we find that expression of the metallothionein (MT) gene family,also linked by GWASs to diabetes,is significantly downregulated in CDKAL1(-/-) cells that have been differentiated to insulin-expressing pancreatic beta-like cells. Forced MT1E expression rescues both hypersensitivity of CDKAL1 mutant cells to glycolipotoxicity and pancreatic beta-cell dysfunction in vitro and in vivo. MT1E functions at least in part through relief of ER stress. This study establishes an isogenic hESC-based platform to study the interaction of GWAS-identified diabetes gene variants and illuminate the molecular network impacting disease progression. View Publication

Human pluripotent stem cells (HPSCs) cultured in conditions that maintain pluripotency via FGF and TGFβ signaling have been described as being in a primed state. These cells have been shown to exhibit characteristics more closely related to mouse epiblast-derived stem cells than to so called naïve mouse PSCs said to possess a more ground state pluripotency that mimics the early mouse embryo inner cell mass. Initial attempts to create culture conditions favorable for generation of naïve HPSCs from primed HPSCs has required the use of mouse embryonic fibroblasts as a feeder layer to support this transition. A protocol for the routine derivation and maintenance of naïve HPSCs in completely defined conditions is highly desirable for stem cell researchers to enhance the study and clinical translation of naïve HPSCs. Here we describe a standard protocol for transitioning primed HPSCs to a naïve state using commercial RSet media and xeno-free recombinant vitronectin. View Publication

Recent reports have documented the differentiation of human pluripotent stem cells toward the skeletal myogenic lineage using transgene- and cell purification-free approaches. Although these protocols generate myocytes,they have not demonstrated scalability,safety,and in vivo engraftment,which are key aspects for their future clinical application. Here we recapitulate one prominent protocol,and show that it gives rise to a heterogeneous cell population containing myocytes and other cell types. Upon transplantation,the majority of human donor cells could not contribute to myofiber formation. As a proof-of-principle,we incorporated the inducible PAX7 lentiviral system into this protocol,which then enabled scalable expansion of a homogeneous population of skeletal myogenic progenitors capable of forming myofibers in vivo. Our findings demonstrate the methods for scalable expansion of PAX7(+) myogenic progenitors and their purification are critical for practical application to cell replacement treatment of muscle degenerative diseases. View Publication

Microglia,the immune cells of the brain,are crucial to proper development and maintenance of the CNS,and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology,we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes,expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca(2+) transients,whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines. View Publication

Recent studies using defined transcription factors to convert skin fibroblasts into chondrocytes have raised the question of whether osteo-chondroprogenitors expressing SOX9 and RUNX2 could also be generated during the course of the reprogramming process. Here,we demonstrated that doxycycline-inducible expression of reprogramming factors (KLF4 [K] and c-MYC [M]) for 6 days were sufficient to convert murine fibroblasts into SOX9(+)/RUNX2(+) cellular aggregates and together with SOX9 (S) promoted the conversion efficiency when cultured in a defined stem cell medium,mTeSR. KMS-reprogrammed cells possess gene expression profiles akin to those of native osteo-chondroprogenitors with elevated osteogenic properties and can differentiate into osteoblasts and chondrocytes in vitro,but form bone tissue upon transplantation under the skin and in the fracture site of mouse tibia. Altogether,we provide a reprogramming strategy to enable efficient derivation of osteo-chondrogenic cells that may hold promise for cell replacement therapy not limited to cartilage but also for bone tissues. View Publication

Deubiquitinating enzymes may play a major regulatory role in pluripotent stem cells (PSCs) but few studies have investigated this topic. Within this family of enzymes,we found that the ubiquitin specific peptidase,USP44,is highly expressed in embryonic stem cells,induced PSCs and testes as compared to differentiated progenies and somatic organs. Analysis by qPCR and 5'RACE showed that alternate promoters are responsible for expression in PSCs and organs. We noticed 7 regions of transcription initiation,some of them with cell- or tissue-specific activity. Close analysis showed that one of the promoters involved in stem cell and testis-specific activity is differentially regulated in those tissues. At the epigenetic level,USP44 transcription was correlated with DNA methylation of a CpG island close to the main promoter region. These data imply a complex picture where regulating factors like OCT4 may interact with other epigenetic mechanisms to regulate USP44 expression in PSCs and testes. View Publication

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens,or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here,to yield functional human haematopoietic stem cells,we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG,HOXA5,HOXA9,HOXA10,LCOR,RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid,B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. View Publication

Gerstmann-Sträussler-Scheinker (GSS) syndrome is a fatal autosomal dominant neurodegenerative prionopathy clinically characterized by ataxia,spastic paraparesis,extrapyramidal signs and dementia. In some GSS familiar cases carrying point mutations in the PRNP gene,patients also showed comorbid tauopathy leading to mixed pathologies. In this study we developed an induced pluripotent stem (iPS) cell model derived from fibroblasts of a GSS patient harboring the Y218N PRNP mutation,as well as an age-matched healthy control. This particular PRNP mutation is unique with very few described cases. One of the cases presented neurofibrillary degeneration with relevant Tau hyperphosphorylation. Y218N iPS-derived cultures showed relevant astrogliosis,increased phospho-Tau,altered microtubule-associated transport and cell death. However,they failed to generate proteinase K-resistant prion. In this study we set out to test,for the first time,whether iPS cell-derived neurons could be used to investigate the appearance of disease-related phenotypes (i.e,tauopathy) identified in the GSS patient. View Publication

It has been postulated that during human fetal development,all cells of the lung epithelium derive from embryonic,endodermal,NK2 homeobox 1-expressing (NKX2-1+) precursor cells. However,this hypothesis has not been formally tested owing to an inability to purify or track these progenitors for detailed characterization. Here we have engineered and developmentally differentiated NKX2-1GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate human primordial lung progenitors that express NKX2-1 but are initially devoid of differentiated lung lineage markers. After sorting to purity,these primordial lung progenitors exhibited lung epithelial maturation. In the absence of mesenchymal coculture support,this NKX2-1+ population was able to generate epithelial-only spheroids in defined 3D cultures. Alternatively,when recombined with fetal mouse lung mesenchyme,the cells recapitulated epithelial-mesenchymal developing lung interactions. We imaged these progenitors in real time and performed time-series global transcriptomic profiling and single-cell RNA sequencing as they moved through the earliest moments of lung lineage specification. The profiles indicated that evolutionarily conserved,stage-dependent gene signatures of early lung development are expressed in primordial human lung progenitors and revealed a CD47hiCD26lo cell surface phenotype that allows their prospective isolation from untargeted,patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine. View Publication

The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair,underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here,we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines,including a WT1-2A-eGFP knock-in reporter line,under chemically-defined,xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells,resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo,as determined by morphological and functional assays,including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies. View Publication