2d-3d-human-induced-pluripotent-stem-cell-based-models-to-dissect

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 ...

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 (IP), detected in HEK293 cells, fibroblasts, iPSCs and differentiating neurons lacking MINPP1. In mutant cells, higher IP 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 IP-mediated chelation on Pontocerebellar Hypoplasia disease pathology and thereby highlight the critical role of MINPP1 in the regulation of human brain development and homeostasis.

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

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.

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

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

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.

Article	Year
MINPP1 prevents intracellular accumulation of the chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia. Nature communications\| PubMed ID: 33257696	2020
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-encod... Stem cell research\| PubMed ID: 32585588	2020
Generation of an iPSC line (IMAGINi022-A) from a patient carrying a SOX10 missense mutation and presenting with deafness, depigmentation and progressive neurological impairment. Stem cell research\| PubMed ID: 32795927	2020
Generation of two induced pluripotent stem cell lines IMAGINi004-A and IMAGINi005-A from healthy donors. Stem cell research\| PubMed ID: 32866896	2020
Generation of an iPSC line (IMAGINi011-A) from a patient carrying a STING mutation. Stem cell research\| PubMed ID: 33340797	2020

Nathalie Lefort

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