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Patient-Specific iPSC-Derived Endothelial Cells Provide Long-Term Phenotypic Correction of Hemophilia A.

ABSTRACT: We generated patient-specific disease-free induced pluripotent stem cells (iPSCs) from peripheral blood CD34+ cells and differentiated them into functional endothelial cells (ECs) secreting factor VIII (FVIII) for gene and cell therapy approaches to cure hemophilia A (HA), an X-linked bleeding disorder caused by F8 mutations. iPSCs were transduced with a lentiviral vector carrying FVIII transgene driven by an endothelial-specific promoter (VEC) and differentiated into bona fide ECs using an optimized protocol. FVIII-expressing ECs were intraportally transplanted in monocrotaline-conditioned non-obese diabetic (NOD) severe combined immune-deficient (scid)-IL2r? null HA mice generating a chimeric liver with functional human ECs. Transplanted cells engrafted and proliferated in the liver along sinusoids, in the long term showed stable therapeutic FVIII activity (6%). These results demonstrate that the hemophilic phenotype can be rescued by transplantation of ECs derived from HA FVIII-corrected iPSCs, confirming the feasibility of cell-reprogramming strategy in patient-derived cells as an approach for HA gene and cell therapy.

SUBMITTER: Olgasi C

PROVIDER: S-EPMC6294075 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Patient-Specific iPSC-Derived Endothelial Cells Provide Long-Term Phenotypic Correction of Hemophilia A.

Olgasi Cristina C Talmon Maria M Merlin Simone S Cucci Alessia A Richaud-Patin Yvonne Y Ranaldo Gabriella G Colangelo Donato D Di Scipio Federica F Berta Giovanni N GN Borsotti Chiara C Valeri Federica F Faraldi Francesco F Prat Maria M Messina Maria M Schinco Piercarla P Lombardo Angelo A Raya Angel A Follenzi Antonia A

Stem cell reports 20181108 6

We generated patient-specific disease-free induced pluripotent stem cells (iPSCs) from peripheral blood CD34+ cells and differentiated them into functional endothelial cells (ECs) secreting factor VIII (FVIII) for gene and cell therapy approaches to cure hemophilia A (HA), an X-linked bleeding disorder caused by F8 mutations. iPSCs were transduced with a lentiviral vector carrying FVIII transgene driven by an endothelial-specific promoter (VEC) and differentiated into bona fide ECs using an opti ...[more]

PMID: 30416049

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Project description:BACKGROUND:Pericytes of the blood-brain barrier (BBB) are embedded within basement membrane between brain microvascular endothelial cells (BMECs) and astrocyte end-feet. Despite the direct cell-cell contact observed in vivo, most in vitro BBB models introduce an artificial membrane that separates pericytes from BMECs. In this study, we investigated the effects of pericytes on BMEC barrier function across a range of in vitro platforms with varied spatial orientations and levels of cell-cell contact. METHODS:We differentiated RFP-pericytes and GFP-BMECs from hiPSCs and monitored transendothelial electrical resistance (TEER) across BMECs on transwell inserts while pericytes were either directly co-cultured on the membrane, indirectly co-cultured in the basolateral chamber, or embedded in a collagen I gel formed on the transwell membrane. We then incorporated pericytes into a tissue-engineered microvessel model of the BBB and measured pericyte motility and microvessel permeability. RESULTS:We found that BMEC monolayers did not require co-culture with pericytes to achieve physiological TEER values (> 1500 Ω cm2). However, under stressed conditions where TEER values for BMEC monolayers were reduced, indirectly co-cultured hiPSC-derived pericytes restored optimal TEER. Conversely, directly co-cultured pericytes resulted in a decrease in TEER by interfering with BMEC monolayer continuity. In the microvessel model, we observed direct pericyte-BMEC contact, abluminal pericyte localization, and physiologically-low Lucifer yellow permeability comparable to that of BMEC microvessels. In addition, pericyte motility decreased during the first 48 h of co-culture, suggesting progression towards pericyte stabilization. CONCLUSIONS:We demonstrated that monocultured BMECs do not require co-culture to achieve physiological TEER, but that suboptimal TEER in stressed monolayers can be increased through co-culture with hiPSC-derived pericytes or conditioned media. We also developed the first BBB microvessel model using exclusively hiPSC-derived BMECs and pericytes, which could be used to examine vascular dysfunction in the human CNS.

Dataset Information

Patient-Specific iPSC-Derived Endothelial Cells Provide Long-Term Phenotypic Correction of Hemophilia A.

Publications

Patient-Specific iPSC-Derived Endothelial Cells Provide Long-Term Phenotypic Correction of Hemophilia A.

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