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.

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.

FLASH Irradiation Spares Lung Progenitor Cells and Limits the Incidence of Radio-induced Senescence.

PURPOSE: One of the main limitations to anticancer radiotherapy lies in irreversible damage to healthy tissues located within the radiation field. "FLASH" irradiation at very high dose-rate is a new treatment modality that has been reported to specifically spare normal tissue from late radiation-induced toxicity in animal models and therefore could be a promising strategy to reduce treatment toxicity. EXPERIMENTAL DESIGN: Lung responses to FLASH irradiation were investigated by qPCR, single-cell RNA sequencing (sc-RNA-Seq), and histologic methods during the acute wound healing phase as well as at late stages using C57BL/6J wild-type and Terc-/- mice exposed to bilateral thorax irradiation as well as human lung cells grown in vitro. RESULTS: In vitro studies gave evidence of a reduced level of DNA damage and induced lethality at the advantage of FLASH. In mouse lung, sc-RNA-seq and the monitoring of proliferating cells revealed that FLASH minimized the induction of proinflammatory genes and reduced the proliferation of progenitor cells after injury. At late stages, FLASH-irradiated lungs presented less persistent DNA damage and senescent cells than after CONV exposure, suggesting a higher potential for lung regeneration with FLASH. Consistent with this hypothesis, the beneficial effect of FLASH was lost in Terc-/- mice harboring critically short telomeres and lack of telomerase activity. CONCLUSIONS: The results suggest that, compared with conventional radiotherapy, FLASH minimizes DNA damage in normal cells, spares lung progenitor cells from excessive damage, and reduces the risk of replicative senescence.

The PACAP-regulated gene selenoprotein T is abundantly expressed in mouse and human ß-cells and its targeted inactivation impairs glucose tolerance.

Selenoproteins are involved in the regulation of redox status, which affects several cellular processes, including cell survival and homeostasis. Considerable interest has arisen recently concerning the role of selenoproteins in the regulation of glucose metabolism. Here, we found that selenoprotein T (SelT), a new thioredoxin-like protein of the endoplasmic reticulum, is present at high levels in human and mouse pancreas as revealed by immunofluorescence and quantitative PCR. Confocal immunohistochemistry studies revealed that SelT is mostly confined to insulin- and somatostatin-producing cells in mouse and human islets. To elucidate the role of SelT in ß-cells, we generated, using a Cre-Lox strategy, a conditional pancreatic ß-cell SelT-knockout C57BL/6J mice (SelT-insKO) in which SelT gene disruption is under the control of the rat insulin promoter Cre gene. Glucose administration revealed that male SelT-insKO mice display impaired glucose tolerance. Although insulin sensitivity was not modified in the mutant mice, the ratio of glucose to insulin was significantly higher in the SelT-insKO mice compared with wild-type littermates, pointing to a deficit in insulin production/secretion in mutant mice. In addition, morphometric analysis showed that islets from SelT-insKO mice were smaller and that their number was significantly increased compared with islets from their wild-type littermates. Finally, we found that SelT is up-regulated by pituitary adenylate cyclase-activating polypeptide (PACAP) in ß-pancreatic cells and that SelT could act by facilitating a feed-forward mechanism to potentiate insulin secretion induced by the neuropeptide. Our findings are the first to show that the PACAP-regulated SelT is localized in pancreatic ß- and δ-cells and is involved in the control of glucose homeostasis.