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Efficient generation, purification, and expansion of CD34(+) hematopoietic progenitor cells from nonhuman primate-induced pluripotent stem cells.

ABSTRACT: Induced pluripotent stem cell (iPSC) therapeutics are a promising treatment for genetic and infectious diseases. To assess engraftment, risk of neoplastic formation, and therapeutic benefit in an autologous setting, testing iPSC therapeutics in an appropriate model, such as the pigtail macaque (Macaca nemestrina; Mn), is crucial. Here, we developed a chemically defined, scalable, and reproducible specification protocol with bone morphogenetic protein 4, prostaglandin-E2 (PGE2), and StemRegenin 1 (SR1) for hematopoietic differentiation of Mn iPSCs. Sequential coculture with bone morphogenetic protein 4, PGE2, and SR1 led to robust Mn iPSC hematopoietic progenitor cell formation. The combination of PGE2 and SR1 increased CD34(+)CD38(-)Thy1(+)CD45RA(-)CD49f(+) cell yield by 6-fold. CD34(+)CD38(-)Thy1(+)CD45RA(-)CD49f(+) cells isolated on the basis of CD34 expression and cultured in SR1 expanded 3-fold and maintained this long-term repopulating HSC phenotype. Purified CD34(high) cells exhibited 4-fold greater hematopoietic colony-forming potential compared with unsorted hematopoietic progenitors and had bilineage differentiation potential. On the basis of these studies, we calculated the cell yields that must be achieved at each stage to meet a threshold CD34(+) cell dose that is required for engraftment in the pigtail macaque. Our protocol will support scale-up and testing of iPSC-derived CD34(high) cell therapies in a clinically relevant nonhuman primate model.

SUBMITTER: Gori JL

PROVIDER: S-EPMC3460690 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Efficient generation, purification, and expansion of CD34(+) hematopoietic progenitor cells from nonhuman primate-induced pluripotent stem cells.

Gori Jennifer L JL Chandrasekaran Devikha D Kowalski John P JP Adair Jennifer E JE Beard Brian C BC D'Souza Sunita L SL Kiem Hans-Peter HP

Blood 20120816 13

Induced pluripotent stem cell (iPSC) therapeutics are a promising treatment for genetic and infectious diseases. To assess engraftment, risk of neoplastic formation, and therapeutic benefit in an autologous setting, testing iPSC therapeutics in an appropriate model, such as the pigtail macaque (Macaca nemestrina; Mn), is crucial. Here, we developed a chemically defined, scalable, and reproducible specification protocol with bone morphogenetic protein 4, prostaglandin-E2 (PGE2), and StemRegenin 1 ...[more]

PMID: 22898598

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Project description:BackgroundDifferentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells.Methodology/principal findingsESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34(+) cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34(+) cells. ESC-derived CD34(+) cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34(-) cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult ?-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors.Conclusions/significanceShort-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature blood cell types. However, additional factors have yet to be identified to allow their differentiation into definitive erythroid cultures.

Dataset Information

Efficient generation, purification, and expansion of CD34(+) hematopoietic progenitor cells from nonhuman primate-induced pluripotent stem cells.

Publications

Efficient generation, purification, and expansion of CD34(+) hematopoietic progenitor cells from nonhuman primate-induced pluripotent stem cells.

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