A cost-effective and efficient reprogramming platform for large-scale production of integration-free human induced pluripotent stem cells in chemically defined culture

Summary Mutations in CEP290, a transition zone protein in primary cilia, cause diverse ciliopathies, including Leber congenital amaurosis (LCA) and Joubert syndrome related disorders (JSRD). We examined cilia biogenesis and function in cells derived from CEP290-LCA and CEP290-JSRD patients. CEP290 protein was reduced in LCA fibroblasts with no detectable impact on cilia; however, optic cups derived from iPSCs of CEP290-LCA patients displayed less-developed photoreceptor cilia. Lack of CEP290 in JSRD fibroblasts resulted in abnormal cilia and decreased ciliogenesis. We observed selectively reduced localization of ADCY3 and ARL13B. Notably, Hedgehog signaling was augmented in CEP290-JSRD because of enhanced ciliary transport of Smoothened and GPR161. These results demonstrate a direct correlation between the extent of ciliogenesis defect(s) in fibroblasts and photoreceptors with phenotypic severity in JSRD and LCA, respectively, and strengthen the role of CEP290 as a selective ciliary gatekeeper for transport of signaling molecules in and out of the cilium.

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“…Fibroblasts were reprogrammed to iPSCs using integration-free Sendai virus as previously described(Beers et al, 2015). …”

Section: Methodsmentioning

confidence: 99%

In Vitro Modeling Using Ciliopathy-Patient-Derived Cells Reveals Distinct Cilia Dysfunctions Caused by CEP290 Mutations

et al. 2017

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“…Peripheral blood CD34 + CD38cells mobilized from healthy donors were reprogrammed into iPSCs (MCND-TENS2 line) using the integration-free CytoTune 2.0 Sendai virus reprogramming kit (A16517, Thermo Fisher Scientific) following the method previously reported 30,31 .…”

Section: Generation and Culture Ipscsmentioning

confidence: 99%

Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system

Villalobos

Chen

Mora

et al. 2019

Preprint

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One of the most promising objectives of clinical hematology is to derive engraftable autologous hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (iPSCs). Progress in translating iPSC technologies to the clinic relies on the availability of scalable differentiation methodologies. In this study, human iPSCs were differentiated for 21 days using STEMdiff™, a monolayer-based approach for hematopoietic differentiation of human iPSCs that requires no replating, co-culture or embryoid body formation. Both monolayer and suspension cells were functionally characterized throughout differentiation. In the supernatant fraction, an early transient population of primitive CD235a+ erythroid cells first emerged, followed by hematopoietic progenitors with multilineage differentiation activity in vitro but no long-term engraftment potential in vivo. In later stages of differentiation, a nearly exclusive production of definitive erythroid progenitors was observed. In the adherent monolayer, we identified a prevalent population of mesenchymal stromal cells and limited arterial vascular endothelium (VE), suggesting that the cellular constitution of the monolayer may be inadequate to support the generation of HSCs with durable repopulating potential. Quantitative modulation of WNT/β-catenin and activin/nodal/TGFβ signaling pathways with CHIR/SB molecules during differentiation enhanced formation of arterial VE, definitive multilineage and erythroid progenitors, but was insufficient to orchestrate the generation of engrafting HSCs. Overall, STEMdiff™ provides a clinically-relevant and readily adaptable platform for the generation of erythroid and multilineage hematopoietic progenitors from human pluripotent stem cells.HighlightsRobust, scalable and clinically-relevant monolayer-based culture system for hematopoietic differentiation of human iPSCs.Successive emergence of primitive erythroid cells, definitive multilineage HSPCs and erythroid progenitors in the culture supernatant.Abundant mesenchymal cells and limited arterial vascular endothelium in the culture monolayer.CHIR/SB molecules increase arterial vascular endothelium formation, suppress primitive hematopoiesis and promote definitive multilineage and erythroid progenitors.

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“…It was reported that one kit might apply to 24-48 blood samples (20, 000 cells each). The viral activity was maintained close to 80% activity of its original stock solution after 1 freeze and thaw cycle, and >50% activity even after 3 cycles [34]. This allows for thawing Sendai virus and preserving it in small aliquoted frozen stocks, which can greatly decrease the cost of reprogramming.…”

Section: Discussionmentioning

confidence: 94%

Efficient Generation of Non-Integration and Feeder-Free Induced Pluripotent Stem Cells from Human Peripheral Blood Cells by Sendai Virus

Wang²

2018

Cell Physiol Biochem

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Background/Aims: Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, disease modeling, and drug development. Thus, generation of non-integration and feeder-free iPSCs is highly desirable for clinical applications. Peripheral blood mononuclear cells (PBMCs) are an attractive resource for cell reprogramming because of their properties of easy accessibility and the limited invasiveness of blood collection. However, derivation of iPSCs is technically demanding due to the low reprogramming efficiency and nonadherent features of PBMCs. Methods: iPSCs were generated from PBMCs using non-integrative Sendai viruses carrying the reprogramming factors Oct4, Sox2, Klf4, and cMyc. The derived iPSCs were fully characterized at the levels of gene and protein, and then they were transplanted into immunocompromised mice for evaluation of in vivo differentiation potential. Three types of extracellular substrates (Geltrex, vitronectin, and rhLaminn-521) were tested for their influences on cell reprogramming under feeder-free conditions. We also sought to establish approaches to efficient cell recovery post-thaw and single cell passaging of iPSCs employing Rock inhibitors. Results: iPSCs were efficiently generated from PBMCs under feeder-free conditions. The derived iPSCs proved to be pluripotent and transgene-free. Furthermore, they demonstrated multi-lineage differentiation potentials when transplanted into immunocompromised mice. Among the three substrates, Geltrex and rhLaminin-521 could effectively support the initial cell reprogramming process, but vitronectin failed. However, the vitronectin, similar to Geltrex and rhLaminin-521, could effectively maintain cell growth and expansion of passaged iPSCs. In addition, RevitaCell supplement (RVC) was more potent on cell recovery post-thaw than Y-27632. And RVC and Y-27632 could significantly increase the cell survival when the cells were passaged in single cells, and they showed comparable effectiveness on cell recovery. Conclusion: We have successfully derived non-integration and feeder-free human iPSCs from peripheral blood cells, and established effective strategies for efficient cell recovery and single cell passaging. This study will pave the way to the derivation of clinical-grade human iPSCs for future clinical applications.

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A cost-effective and efficient reprogramming platform for large-scale production of integration-free human induced pluripotent stem cells in chemically defined culture

Cited by 99 publications

References 13 publications

In Vitro Modeling Using Ciliopathy-Patient-Derived Cells Reveals Distinct Cilia Dysfunctions Caused by CEP290 Mutations

In Vitro Modeling Using Ciliopathy-Patient-Derived Cells Reveals Distinct Cilia Dysfunctions Caused by CEP290 Mutations

Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system

Efficient Generation of Non-Integration and Feeder-Free Induced Pluripotent Stem Cells from Human Peripheral Blood Cells by Sendai Virus

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