Efficient generation, purification, and expansion of CD34(+) hematopoietic progenitor cells from nonhuman primate-induced pluripotent stem cells.
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.
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
Jennifer L
JL
Gori
Devikha
D
Chandrasekaran
John P
JP
Kowalski
Jennifer E
JE
Adair
Brian C
BC
Beard
Sunita L
SL
D'Souza
Hans-Peter
HP
Kiem
D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism*; D015153:Blotting, Western; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D015232:Dinoprostone / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism; D008251:Macaca; D022423:Myeloid Cells / Q000166:cytology / Q000378:metabolism; D011687:Purines / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053586:Virus Internalization; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism
D018952:Antigens, CD34; D055415:Bone Morphogenetic Protein 4; D011687:Purines; D012333:RNA, Messenger; C552240:StemRegenin 1; D051153:Wnt Proteins; D015232:Dinoprostone