Differentiation of haemopoietic stem cells and progenitor cells from human pluripotent stem cells

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• 作者：Dr Jennifer Easterbrook ; MBChB^a ; ^{Jennifer.Easterbrook@ed.ac.uk" class="auth_mail" title="E-mail the corresponding author} ; Prof Lesley Forrester ; PhD^a ; Prof Alexander Medvinsky ; PhD^a
• 刊名：The Lancet
• 出版年：2016
• 出版时间：25 February 2016
• 年：2016
• 卷：387
• 期：supp_S1
• 页码：S39
• 全文大小：50 K

文摘

Induced pluripotent stem cells hold great promise as an unlimited source of clinical grade immunologically matched haemopoietic stem cells (HSCs) for transplantation. However, there are currently no robust, reproducible differentiation protocols for the production of engraftable HSCs. We aimed to characterise the production of haemopoietic progenitor cells (HPCs) and HSCs during differentiation of human embryonic stem cells and induced pluripotent stem cells.

Methods

We used a serum-free, feeder-free, good manufacturing practice-compliant differentiation protocol established by the Novosang Consortium for the production of red blood cells for transfusion. Cell phenotype was assessed by flow cytometry for markers of HPCs and HSCs. Cell function was assessed in vitro by the colony forming units in culture (CFU-C) assay and in vivo with a long-term transplantation assay.

Findings

After preliminary studies, the work focused on days 6, 8, and 10 of the differentiation protocol. In seven independent experiments, HPCs were reproducibly produced at high levels with over 90% of cells expressing CD43 by day 10. Almost all CD41+ cells coexpressed CD43 at all timepoints (day 10, 65% CD43+CD41+). CFU-Cs emerged in large numbers from day 6 and increased by day 10 (mean 1000 colonies per 10⁵ cells plated [SD 478], six experiments). Cytospin and flow cytometry confirmed production of erythroid, granulocyte and macrophage, macrophage, and mixed colonies. The human embryonic HSC markers—vascular endothelial cadherin and CD45—were coexpressed at low levels (mean 3·6% [SD 2·5], seven experiments). No human engraftment was detected when cells were injected into immune-deficient NSG (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ) mice.

Interpretation

This differentiation protocol reproducibly gave a high yield of HPCs. However, there was no evidence of engraftable HSCs being produced. This finding could indicate inadequate extrinsic signalling during differentiation. Further work will therefore focus on co-culturing human pluripotent stem cells with human stromal cells in an attempt to mimic the embryonic HSC niche.

Funding

Wellcome Trust.