Mechano-osmotic signals control chromatin state and fate transitions in pluripotent stem cells.

Acquisition of specific cell shapes and morphologies is a central component of cell fate transitions. Although signaling circuits and gene regulatory networks that regulate pluripotent stem cell differentiation have been intensely studied, how these networks are integrated in space and time with morphological transitions and mechanical deformations to control state transitions remains a fundamental open question. Here, we focus on two distinct models of pluripotency, primed pluripotent stem cells and pre-implantation inner cell mass cells of human embryos to discover that cell fate transitions associate with rapid changes in nuclear shape and volume which collectively alter the nuclear mechanophenotype. Mechanistic studies in human induced pluripotent stem cells further reveal that these phenotypical changes and the associated active fluctuations of the nuclear envelope arise from growth factor signaling-controlled changes in chromatin mechanics and cytoskeletal confinement. These collective mechano-osmotic changes trigger global transcriptional repression and a condensation-prone environment that primes chromatin for a cell fate transition by attenuating repression of differentiation genes. However, while this mechano-osmotic chromatin priming has the potential to accelerate fate transitions and differentiation, sustained biochemical signals are required for robust induction of specific lineages. Our findings uncover a critical mechanochemical feedback mechanism that integrates nuclear mechanics, shape and volume with biochemical signaling and chromatin state to control cell fate transition dynamics.

Six induced pluripotent stem cell lines from fibroblasts of individuals with CLN3-related conditions.

Primary fibroblasts from six individuals with CLN3-related conditions were used to generate induced pluripotent stem cell (iPSC) lines CHDTRi001-B, CHDTRi002-B, CHDTRi003-A, CHDTRi004-B, CHDTRi005-A, and CHDTRi006-E through the expression of four reprogramming factors: human OCT3/4, KLF4, SOX2, and c-MYC. The iPSC lines were characterized to confirm their pluripotency via immunocytochemistry, flow cytometry, and teratoma formation. Genomic stability, cell line identity, and CLN3 genotype were confirmed. These iPSC lines may be used as participant-derived experimental models for further investigation of CLN3, a rare, fatal, pediatric, blindness and neurodegenerative lysosomal disorder with no cure.

Establishment of a human induced pluripotent stem cell line (NIHTVBi031-A) derived from a COPA syndrome patient with a heterozygous p.Ala239Pro mutation.

We have successfully generated human induced pluripotent stem cells (hiPSC) from peripheral blood mononuclear cells (PBMCs) of a patient with COPA Syndrome. The patient, a 6 year old Caucasian male, has a spontaneous de novo missense mutation that replaced alanine with proline in the COPA gene. This paper confirms the differentiation potential of the hiPSC line, the presence of the p.Ala239Pro mutation, and the expression of typical pluripotency markers within the hiPSC line. The hiPSC line is ready for use as a cellular model of COPA Syndrome.

Human induced pluripotent stem cells (NIHTVBi029-A and NIHTVBi030-A) generated from two patients with a heterozygous mutation in the CDC42 gene.

Induced pluripotent stem cells (iPSCs) were successfully generated from peripheral blood mononuclear cells obtained from two patients with a heterozygous mutation in the CDC42 gene. Both iPSC lines expressed pluripotency markers, differentiated into the three germ layers in vitro, showed normal karyotypes, and retained the disease-causing mutation. Created iPSC lines and their differentiated derivatives may be of interest in the study of the physiology of disease mechanisms and therapy.

Long-term engraftment and maturation of autologous iPSC-derived cardiomyocytes in two rhesus macaques.

Cellular therapies with cardiomyocytes produced from induced pluripotent stem cells (iPSC-CMs) offer a potential route to cardiac regeneration as a treatment for chronic ischemic heart disease. Here, we report successful long-term engraftment and in vivo maturation of autologous iPSC-CMs in two rhesus macaques with small, subclinical chronic myocardial infarctions, all without immunosuppression. Longitudinal positron emission tomography imaging using the sodium/iodide symporter (NIS) reporter gene revealed stable grafts for over 6 and 12 months, with no teratoma formation. Histological analyses suggested capability of the transplanted iPSC-CMs to mature and integrate with endogenous myocardium, with no sign of immune cell infiltration or rejection. By contrast, allogeneic iPSC-CMs were rejected within 8 weeks of transplantation. This study provides the longest-term safety and maturation data to date in any large animal model, addresses concerns regarding neoantigen immunoreactivity of autologous iPSC therapies, and suggests that autologous iPSC-CMs would similarly engraft and mature in human hearts.

Generation of an Alagille Syndrome (ALGS) patient-derived induced pluripotent stem cell line (TRNDi036-A) carrying a heterozygous mutation (p.Cys693*) in the JAG1 gene.

Alagille syndrome (ALGS) is an autosomal dominant, multisystemic disorder due to haploinsufficiency in JAG1 or less frequently, mutations in NOTCH2. The disease has been difficult to diagnose and treat due to variable expression. The generation of this iPSC line (TRNDi036-A) carrying a heterozygous mutation (p.Cys693*) in the JAG1 gene provides a means of studying the disease and developing novel therapeutics towards patient treatment.

Generation of an Alagille syndrome (ALGS) patient-derived induced pluripotent stem cell line (TRNDi032-A) carrying a heterozygous mutation (p.Cys682Leufs*7) in the JAG1 gene.

Alagille syndrome (ALGS) is an autosomal dominant, multisystemic disorder due to haploinsufficiency in either the JAG1 gene (ALGS type 1) or the NOTCH2 gene (ALGS type 2). The disease has been difficult to diagnose and treat due to its muti-system clinical presentation, variable expressivity, and prenatal onset for some of the features. The generation of this iPSC line (TRNDi032-A) carrying a heterozygous mutation, p.Cys682Leufs*7 (c.2044dup), in the JAG1 gene provides a means of studying the disease and developing novel therapeutics towards patient treatment.

A Protocol for Culture and Characterization of Human Induced Pluripotent Stem Cells After Induction.

Human induced pluripotent stem cells (hiPSCs) are characterized by unlimited self-renewal and the capability to differentiate into all three germ layers, with the potential to further differentiate into all types of cells and tissues. Human iPSCs retain all genetic information from their original donors and can be developed into disease models to study disease pathophysiology, identify disease phenotypes and biomarkers, and evaluate therapeutic efficacy and toxicity for drug development. Human iPSCs can also be used to develop cell therapies and regenerative medicine. In the last decade, the technologies for hiPSC generation and differentiation have advanced rapidly. Human iPSC culture and propagation are tedious and require careful handling. High-quality hiPSCs are necessary for downstream applications. The methods, techniques, and skills for hiPSC maintenance and characterization are very different from those for immortalized cell lines. It can be a challenge for new laboratory staff, and sometimes even for experienced staff, to properly culture and maintain the high quality of these cells. Here, we describe a comprehensive set of protocols for hiPSC propagation under chemically defined and feeder-free culture conditions. These step-by-step protocols describe in detail all the reagents and experimental procedures needed to culture hiPSCs. The protocols also describe experimental methods for hiPSC characterization, including immunofluorescence staining and flow cytometric analysis with a panel of pluripotency markers, a teratoma formation assay for validation of in vivo pluripotency, and detection of Sendai virus to ensure elimination of the viral vectors. These protocols have been successfully used in our laboratory for hiPSC expansion and propagation, and this article provide a useful reference guide for laboratory staff to work on hiPSC culture. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Propagation and cryopreservation of hiPSC cultures Basic Protocol 2: Recovery of cryopreserved hiPSCs Basic Protocol 3: Validation of pluripotency markers via immunocytochemical analysis Alternate Protocol: Determination of the expression of pluripotency markers via flow cytometry analysis Basic Protocol 4: Assessment of pluripotency via in vivo teratoma formation assay Basic Protocol 5: Confirmation of Sendai viral vector clearance via RT-PCR.

Three induced pluripotent stem cell lines (TRNDi033-A, TRNDi034-A, TRNDi035-A) generated from lymphoblasts of three apparently healthy individuals.

Expanded human lymphoblast cells from three different aged healthy individuals, 8-year-old male, 0-year-old newborn (NB) male, and 26-year-old female, were used to generate induced pluripotent stem cell (iPSC) lines TRNDi033-A, TRNDi034-A and TRNDi035-A, respectively, by exogenous expression of five reprogramming factors, human OCT4, SOX2, KLF4, L-MYC and LIN28. The authenticity of established iPSC lines was confirmed by the expressions of stem cell markers, karyotype analysis, embryoid body formation, and scorecard analysis. These iPSC lines could serve as healthy donor controls that are age and sex matched for the studies involving patient-specific iPSCs.

Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells.

Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. We found that hiPSCs cocultured with stromal cells as spheroids (hematopoietic spheroids [Hp-spheroids]) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lympho-myeloid potential. Moreover, sequential hemato-vascular ontogenesis could also be observed during organoid formation. We demonstrated that organoid-induced HPCs can be differentiated into erythroid cells, macrophages, and T lymphocytes with current maturation protocols. Notably, the Hp-spheroid system can be performed in an autologous and xeno-free manner, thereby improving the feasibility of bulk production of hiPSC-derived HPCs in clinical, therapeutic contexts.

Modulation of WNT, Activin/Nodal, and MAPK Signaling Pathways Increases Arterial Hemogenic Endothelium and Hematopoietic Stem/Progenitor Cell Formation During Human iPSC Differentiation.

Several differentiation protocols enable the emergence of hematopoietic stem and progenitor cells (HSPCs) from human-induced pluripotent stem cells (iPSCs), yet optimized schemes to promote the development of HSPCs with self-renewal, multilineage differentiation, and engraftment potential are lacking. To improve human iPSC differentiation methods, we modulated WNT, Activin/Nodal, and MAPK signaling pathways by stage-specific addition of small-molecule regulators CHIR99021, SB431542, and LY294002, respectively, and measured the impact on hematoendothelial formation in culture. Manipulation of these pathways provided a synergy sufficient to enhance formation of arterial hemogenic endothelium (HE) relative to control culture conditions. Importantly, this approach significantly increased production of human HSPCs with self-renewal and multilineage differentiation properties, as well as phenotypic and molecular evidence of progressive maturation in culture. Together, these findings provide a stepwise improvement in human iPSC differentiation protocols and offer a framework for manipulating intrinsic cellular cues to enable de novo generation of human HSPCs with functionality in vivo.

Modulation of WNT, Activin/Nodal and MAPK Signaling Pathways Increases Arterial Hemogenic Endothelium and Hematopoietic Stem/Progenitor Cell Formation During Human iPSC Differentiation.

Several differentiation protocols enable the emergence of hematopoietic stem and progenitor cells (HSPCs) from human induced pluripotent stem cells (iPSCs), yet optimized schemes to promote the development of HSPCs with self-renewal, multilineage differentiation and engraftment potential are lacking. To improve human iPSC differentiation methods, we modulated WNT, Activin/Nodal and MAPK signaling pathways by stage-specific addition of small molecule regulators CHIR99021, SB431542 and LY294002, respectively, and measured the impact on hematoendothelial formation in culture. Manipulation of these pathways provided a synergy sufficient to enhance formation of arterial hemogenic endothelium (HE) relative to control culture conditions. Importantly, this approach significantly increased production of human HSPCs with self-renewal and multilineage differentiation properties, as well as phenotypic and molecular evidence of progressive maturation in culture. Together, these findings provide a stepwise improvement in human iPSC differentiation protocols and offer a framework for manipulating intrinsic cellular cues to enable de novo generation of human HSPCs with functionality in vivo . Significance Statement: The ability to produce functional HSPCs by differentiation of human iPSCs ex vivo holds enormous potential for cellular therapy of human blood disorders. However, obstacles still thwart translation of this approach to the clinic. In keeping with the prevailing arterial-specification model, we demonstrate that concurrent modulation of WNT, Activin/Nodal and MAPK signaling pathways by stage-specific addition of small molecules during human iPSC differentiation provides a synergy sufficient to promote arterialization of HE and production of HSPCs with features of definitive hematopoiesis. This simple differentiation scheme provides a unique tool for disease modeling, in vitro drug screening and eventual cell therapies.

Transcriptomic Analyses of Brains of RBM8A Conditional Knockout Mice at Different Developmental Stages Reveal Conserved Signaling Pathways Contributing to Neurodevelopmental Diseases.

RNA-binding motif 8A (RBM8A) is a core component of the exon junction complex (EJC) that binds pre-mRNAs and regulates their splicing, transport, translation, and nonsense-mediated decay (NMD). Dysfunction in the core proteins has been linked to several detriments in brain development and neuropsychiatric diseases. To understand the functional role of Rbm8a in brain development, we have generated brain-specific Rbm8a knockout mice and used next-generation RNA-sequencing to identify differentially expressed genes (DEGs) in mice with heterozygous, conditional knockout (cKO) of Rbm8a in the brain at postnatal day 17 (P17) and at embryonic day 12. Additionally, we analyzed enriched gene clusters and signaling pathways within the DEGs. At the P17 time point, between the control and cKO mice, about 251 significant DEGs were identified. At E12, only 25 DEGs were identified in the hindbrain samples. Bioinformatics analyses have revealed many signaling pathways related to the central nervous system (CNS). When E12 and P17 results were compared, three DEGs, Spp1, Gpnmb, and Top2a, appeared to peak at different developmental time points in the Rbm8a cKO mice. Enrichment analyses suggested altered activity in pathways affecting cellular proliferation, differentiation, and survival. The results support the hypothesis that loss of Rbm8a causes decreased cellular proliferation, increased apoptosis, and early differentiation of neuronal subtypes, which may lead ultimately to an altered neuronal subtype composition in the brain.

The endo-lysosomal regulatory protein BLOC1S1 modulates hepatic lysosomal content and lysosomal lipolysis.

BLOC1S1 is a common component of BLOC and BORC multiprotein complexes which play distinct roles in endosome and lysosome biology. Recent human mutations in BLOC1S1 associate with juvenile leukodystrophy. As leukodystrophy is linked to perturbed lysosomal lipid storage we explored whether BLOC1S1 itself modulates this biology. Given the central role of the liver in lipid storage, our investigations were performed in hepatocyte specific liver bloc1s1 knockout (LKO) mice and in human hepatocyte-like lines (HLCs) derived from inducible pluripotential stem cells (iPSCs) from a juvenile leukodystrophy subject's with bloc1s1 mutations and from isogenic corrected iPSCs. Here we show that hepatocyte lipid stores are diminished in parallel with increased lysosomal content, increased lysosomal lipid uptake and lipolysis in LKO mice. The lysosomal lipolysis program was independent of macro- and chaperone-mediated lipophagy but dependent on cellular lysosome content. In parallel, genetic induction of lysosomal biogenesis in a transformed hepatocyte cell line replicated depletion of intracellular lipid stores. Interestingly bloc1s1 mutant and isogenic corrected HLCs both showed normal lysosomal enzyme activity. However, relative to the isogenic corrected HLCs, mutant bloc1s1 HLCs showed reduced lysosomal content and increased lipid storage. Together these data show distinct phenotypes in human mutant HLCs compared to murine knockout cells. At the same time, human blcs1s1 mutation and murine hepatocyte bloc1s1 depletion disrupt lysosome content and the cellular lipid storage. These data support that BLOC1S1 modulates lysosome content and lipid handling independent of autophagy and show that lysosomal lipolysis is dependent on the cellular content of functional lysosomes.

Ligand-free mitochondria-localized mutant AR-induced cytotoxicity in spinal bulbar muscular atrophy.

Spinal bulbar muscular atrophy (SBMA), the first identified CAG-repeat expansion disorder, is an X-linked neuromuscular disorder involving CAG-repeat-expansion mutations in the androgen receptor (AR) gene. We utilized CRISPR-Cas9 gene editing to engineer novel isogenic human induced pluripotent stem cell (hiPSC) models, consisting of isogenic AR knockout, control and disease lines expressing mutant AR with distinct repeat lengths, as well as control and disease lines expressing FLAG-tagged wild-type and mutant AR, respectively. Adapting a small-molecule cocktail-directed approach, we differentiate the isogenic hiPSC models into motor neuron-like cells with a highly enriched population to uncover cell-type-specific mechanisms underlying SBMA and to distinguish gain- from loss-of-function properties of mutant AR in disease motor neurons. We demonstrate that ligand-free mutant AR causes drastic mitochondrial dysfunction in neurites of differentiated disease motor neurons due to gain-of-function mechanisms and such cytotoxicity can be amplified upon ligand (androgens) treatment. We further show that aberrant interaction between ligand-free, mitochondria-localized mutant AR and F-ATP synthase is associated with compromised mitochondrial respiration and multiple other mitochondrial impairments. These findings counter the established notion that androgens are requisite for mutant AR-induced cytotoxicity in SBMA, reveal a compelling mechanistic link between ligand-free mutant AR, F-ATP synthase and mitochondrial dysfunction, and provide innovative insights into motor neuron-specific therapeutic interventions for SBMA.

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

Human induced pluripotent stem cells generated from STING-associated vasculopathy with onset in infancy (SAVI) patients with a heterozygous mutation in the STING gene.

We have successfully created induced pluripotent stem cells (iPSC) from patients carrying a heterozygous mutation in the gene encoding STING. The gain-of-function mutation leads to constitutive activation of STING which leads to the development of the disease STING-associated vasculopathy with onset in infancy (SAVI). The iPSC lines derived from the SAVI patitents are shown to be morphologically and phenotypically normal and have the potential to self renew and differentiate into the three germ layers. These iPSC provide a powerful tools to investigate the role of STING in the regulation of immune responses and vascular renegeration.

Human induced pluripotent stem cells generated from a patient with a homozygous mutation in the Lyn kinase gene.

We have successfully generated induced pluripotent stem cells (iPSC) from dermal fibroblasts of the patient with a germline mutation in the coding region of the LYN kinase gene. This gain of function (GOF) mutation eliminates the inhibitory tyrosine (Y) at the position p.Y508, with an unknown established disease etiology. The iPSC carrying germline mutation in LYN are phenotypically normal, and they have capacity to differentiate toward the three germ layers. These iPSCs are critical for studying this unknown disease etiology and to the further understand the role of Lyn kinases in autoimmune disease.