Generation of IPi001-A/B/C human induced pluripotent stem cell lines from healthy amniotic fluid cells.

Human induced Pluripotent Stem Cells (hiPSCs) represent an invaluable source of primary cells to investigate development, establish cell and disease models, provide material for regenerative medicine and allow more physiological high-content screenings. Here, we generated three healthy hiPSC control lines - IPi001-A/B/C - from primary amniotic fluid cells (AFCs), an infrequently used source of cells, which can be readily obtained from amniocentesis for the prenatal diagnosis of numerous genetic disorders. These AFCs were reprogrammed by non-integrative viral transduction. The resulting hiPSCs displayed normal karyotype and expressed classic pluripotency hallmarks.

Generation and characterization of induced pluripotent stem cell lines from two patients with recessive dystrophic epidermolysis Bullosa.

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic disease responsible for blistering of the skin and mucosa caused by a wide variety of mutations in COL7A1 encoding type VII collagen. We have generated Induced Pluripotent Stem Cells (iPSCs) from two RDEB patients' fibroblasts harboring homozygous recurrent mutations in COL7A1. Their pluripotent state was confirmed by gene and protein expression of stem cell markers OCT4, SOX2, TRA1/60 and SSEA4. Embryoid body formation followed by immunostaining and TaqMan scorecard analysis confirmed the capacity of RDEB iPSCs to differentiate into cell types from the three germ layers in vitro.

Generation of induced pluripotent stem cells (iPSCs) from a microvillus inclusion disease patient with a homozygous missense mutation in UNC45A.

Mutations in UNC45A, a co-chaperone for myosins, were recently found causative of a syndrome combining cholestasis, diarrhea, loss of hearing and bone fragility. We generated induced pluripotent stem cells (iPSCs) from a patient with a homozygous missense mutation in UNC45A. Cells from this patient, which were reprogrammed using integration-free Sendaï virus, have normal karyotype, express pluripotency markers and are able to differentiate into the three germ cell layers.

Engineering 3D micro-compartments for highly efficient and scale-independent expansion of human pluripotent stem cells in bioreactors.

Human pluripotent stem cells (hPSCs) have emerged as the most promising cellular source for cell therapies. To overcome the scale-up limitations of classical 2D culture systems, suspension cultures have been developed to meet the need for large-scale culture in regenerative medicine. Despite constant improvements, current protocols that use microcarriers or generate cell aggregates only achieve moderate amplification performance. Here, guided by reports showing that hPSCs can self-organize in vitro into cysts reminiscent of the epiblast stage in embryo development, we developed a physio-mimetic approach for hPSC culture. We engineered stem cell niche microenvironments inside microfluidics-assisted core-shell microcapsules. We demonstrate that lumenized three-dimensional colonies significantly improve viability and expansion rates while maintaining pluripotency compared to standard hPSC culture platforms such as 2D cultures, microcarriers, and aggregates. By further tuning capsule size and culture conditions, we scale up this method to industrial-scale stirred tank bioreactors and achieve an unprecedented hPSC amplification rate of 277-fold in 6.5 days. In brief, our findings indicate that our 3D culture system offers a suitable strategy both for basic stem cell biology experiments and for clinical applications.

UNC45A deficiency causes microvillus inclusion disease-like phenotype by impairing myosin VB-dependent apical trafficking.

Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development.

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

Generation and characterization of IMAGINi013-A, an induced pluripotent stem cell line generated from a healthy donor.

Human pluripotent stem cells are a powerful tool to study development, to model diseases or as cellular substrates for drug screening. We generated a human induced pluripotent stem cell (hiPSC) line from a healthy control donor. Peripheral blood mononuclear cells (PBMCs) from this donor were reprogrammed using integration-free Sendai virus. This cell line had normal karyotype, expressed pluripotency hallmarks and differentiated into the three primary germ layers.

Generation of an iPSC line (IMAGINi011-A) from a patient carrying a STING mutation.

Mutation in STING1gene, which encodes stimulator of type I IFN gene (STING) leads to its constitutive activation and thereby to a severe vasculopathy and sometimes a lupus-like disease. We generated induced pluripotent stem cells (iPSCs) from a patient carrying a rare heterozygous variant c.463G > A (resulting in a p.V155M substitution) in STING1. Cells from this patient, which were reprogrammed by non-integrative viral transduction, had normal karyotype, expressed pluripotency markers and were able to differentiate into the three germ cell layers.

MINPP1 prevents intracellular accumulation of the chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia.

Inositol polyphosphates are vital metabolic and secondary messengers, involved in diverse cellular functions. Therefore, tight regulation of inositol polyphosphate metabolism is essential for proper cell physiology. Here, we describe an early-onset neurodegenerative syndrome caused by loss-of-function mutations in the multiple inositol-polyphosphate phosphatase 1 gene (MINPP1). Patients are found to have a distinct type of Pontocerebellar Hypoplasia with typical basal ganglia involvement on neuroimaging. We find that patient-derived and genome edited MINPP1-/- induced stem cells exhibit an inefficient neuronal differentiation combined with an increased cell death. MINPP1 deficiency results in an intracellular imbalance of the inositol polyphosphate metabolism. This metabolic defect is characterized by an accumulation of highly phosphorylated inositols, mostly inositol hexakisphosphate (IP6), detected in HEK293 cells, fibroblasts, iPSCs and differentiating neurons lacking MINPP1. In mutant cells, higher IP6 level is expected to be associated with an increased chelation of intracellular cations, such as iron or calcium, resulting in decreased levels of available ions. These data suggest the involvement of IP6-mediated chelation on Pontocerebellar Hypoplasia disease pathology and thereby highlight the critical role of MINPP1 in the regulation of human brain development and homeostasis.

Generation of two induced pluripotent stem cell lines IMAGINi004-A and IMAGINi005-A from healthy donors.

Human pluripotent stem cells offer a limitless source of cells for regenerative medicine. They are also highly valuable as tools to study development and pathologies or as cellular substrates to screen and test new drugs. We generated human induced pluripotent stem cell (hiPSC) lines from two unrelated healthy control donors. Peripheral blood mononuclear cells (PBMCs) from these donors were reprogrammed by non-integrative viral transduction, had normal karyotypes and expressed pluripotency hallmarks.

Generation of an iPSC line (IMAGINi022-A) from a patient carrying a SOX10 missense mutation and presenting with deafness, depigmentation and progressive neurological impairment.

Mutations of SOX10 result in a broad range of phenotypes including Waardenburg syndrome (WS types 2 and 4) that can be found in association with peripheral demyelinating neuropathy and/or central dysmyelinating leukodystrophy. Here, we generated induced pluripotent stem cells (iPSCs) from a patient carrying a de novo heterozygous missense mutation in the SOX10 gene (MIM* 602229, NM006941.3c.523C > G; p.Pro175Ala) presenting with deafness, depigmentation and progressive neurological impairment. Cells were reprogrammed by non-integrative viral transduction from blood sample, have normal karyotype, express pluripotency markers and are able to differentiate into the three germ cell layers.

Generation of an induced pluripotent stem cell (iPSC) line (IMAGINi007) from a patient with steroid-resistant nephrotic syndrome carrying the homozygous p.R138Q mutation in the podocin-encoding NPHS2 gene.

Mutations in the NPHS2 gene, encoding podocin, are responsible for the majority of familial cases of steroid-resistant nephrotic syndrome (SRNS), a rare glomerulopathy that rapidly progresses to end-stage renal disease. We obtained peripheral blood mononuclear cells (PBMCs) from a patient carrying the homozygous c.413G>A substitution (p.R138Q) in NPHS2 gene, which is the most prevalent mutation in the European population. The PBMCs were reprogrammed by non-integrative viral transduction of the Yamanaka's factors. The resulting iPSCs display normal karyotype, express pluripotency hallmarks and are capable of multilineage differentiation, offering a useful tool to study pathological mechanisms of SRNS and perform drug testing.

High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect.

Proper brain development relies highly on protein N-glycosylation to sustain neuronal migration, axon guidance and synaptic physiology. Impairing the N-glycosylation pathway at early steps produces broad neurological symptoms identified in congenital disorders of glycosylation. However, little is known about the molecular mechanisms underlying these defects. We generated a cerebellum specific knockout mouse for Srd5a3, a gene involved in the initiation of N-glycosylation. In addition to motor coordination defects and abnormal granule cell development, Srd5a3 deletion causes mild N-glycosylation impairment without significantly altering ER homeostasis. Using proteomic approaches, we identified that Srd5a3 loss affects a subset of glycoproteins with high N-glycans multiplicity per protein and decreased protein abundance or N-glycosylation level. As IgSF-CAM adhesion proteins are critical for neuron adhesion and highly N-glycosylated, we observed impaired IgSF-CAM-mediated neurite outgrowth and axon guidance in Srd5a3 mutant cerebellum. Our results link high N-glycan multiplicity to fine-tuned neural cell adhesion during mammalian brain development.

Early transcriptional changes linked to naturally occurring Huntington's disease mutations in neural derivatives of human embryonic stem cells.

Huntington's disease (HD) is characterized by a late clinical onset despite ubiquitous expression of the mutant gene at all developmental stages. How mutant huntingtin impacts on signalling pathways in the pre-symptomatic period has remained essentially unexplored in humans due to a lack of appropriate models. Using multiple human embryonic stem cell lines derived from blastocysts diagnosed as carrying the mutant huntingtin gene by pre-implantation genetic diagnosis, we explored early developmental changes in gene expression using differential transcriptomics, combined with gain and loss of function strategies. We demonstrated a down-regulation of the HTT gene itself in HD neural cells and identified three genes, the expression of which differs significantly in HD cells when compared with wild-type controls, namely CHCHD2, TRIM4 and PKIB. Similar dysregulation had been observed previously for CHCDH2 and TRIM4 in blood cells from patients. CHCHD2 is involved in mitochondrial function and PKIB in protein kinase A-dependent pathway regulation, which suggests that these functions may be precociously impacted in HD.

Recurrent genomic instability of chromosome 1q in neural derivatives of human embryonic stem cells.

Human pluripotent stem cells offer a limitless source of cells for regenerative medicine. Neural derivatives of human embryonic stem cells (hESCs) are currently being used for cell therapy in 3 clinical trials. However, hESCs are prone to genomic instability, which could limit their clinical utility. Here, we report that neural differentiation of hESCs systematically produced a neural stem cell population that could be propagated for more than 50 passages without entering senescence; this was true for all 6 hESC lines tested. The apparent spontaneous loss of evolution toward normal senescence of somatic cells was associated with a jumping translocation of chromosome 1q. This chromosomal defect has previously been associated with hematologic malignancies and pediatric brain tumors with poor clinical outcome. Neural stem cells carrying the 1q defect implanted into the brains of rats failed to integrate and expand, whereas normal cells engrafted. Our results call for additional quality controls to be implemented to ensure genomic integrity not only of undifferentiated pluripotent stem cells, but also of hESC derivatives that form cell therapy end products, particularly neural lines.

Human embryonic stem cells and genomic instability.

Owing to their original properties, pluripotent human embryonic stem cells (hESCs) and their progenies are highly valuable not only for regenerative medicine, but also as tools to study development and pathologies or as cellular substrates to screen and test new drugs. However, ensuring their genomic integrity is one important prerequisite for both research and therapeutic applications. Until recently, several studies about the genomic stability of cultured hESCs had described chromosomal or else large genomic alterations detectable with conventional karyotypic methods. In the past year, several laboratories have reported many small genomic alterations, in the megabase-sized range, using more sensitive karyotyping methods, showing that hESCs are prone to acquire focal genomic abnormalities in culture. As these alterations were found to be nonrandom, these findings strongly advocate for high-resolution monitoring of human pluripotent stem cell lines, especially when intended to be used for clinical applications.

Human embryonic stem cells reveal recurrent genomic instability at 20q11.21.

By analyzing five human embryonic stem (hES) cell lines over long-term culture, we identified a recurrent genomic instability in the human genome. An amplification of 2.5-4.6 Mb at 20q11.21, encompassing approximately 23 genes in common, was detected in four cell lines of different origins. This amplification, which has been associated with oncogenic transformation, may provide a selective advantage to hES cells in culture.

Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats.

Substitutive cell therapy using fetal striatal grafts has demonstrated preliminary clinical success in patients with Huntington's disease, but the logistics required for accessing fetal cells preclude its extension to the relevant population of patients. Human embryonic stem (hES) cells theoretically meet this challenge, because they can be expanded indefinitely and differentiated into any cell type. We have designed an in vitro protocol combining substrates, media, and cytokines to push hES cells along the neural lineage, up to postmitotic neurons expressing striatal markers. The therapeutic potential of such hES-derived cells was further substantiated by their in vivo differentiation into striatal neurons following xenotransplantation into adult rats. Our results open the way toward hES cell therapy for Huntington's disease. Long-term proliferation of human neural progenitors leads, however, to xenograft overgrowth in the rat brain, suggesting that the path to the clinic requires a way to switch them off after grafting.