Perivascular stromal cells from primate pluripotent stem cells
a technology of perivascular stromal cells and stem cells, applied in the field of stem cell biology, can solve the problems of limited supply of primate pericytes as cells for cell therapy, limited scalability, heterogeneous and limited scalability,
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example 1
Derivation of Pericytes / Perivascular Stromal Cells from Human Embryonic Stem Cells
[0130]The derivation process for generating pericytes / perivascular stromal cells from human embryonic stem cells was based on a directed differentiation protocol previously used to generate endothelial progenitor cells, described in U.S. patent application Ser. No. 13 / 477,002. We discovered that by altering the cell density during embryoid body (EB) formation and during the culturing of EBs in suspension, the resulting cells can be driven either toward endothelial progenitor cells (low cell density during EB formation and EB suspension culture), or alternatively, toward pericytes / perivascular stromal cells (high cell density during EB formation and EB suspension culture) (FIG. 7).
[0131]The differentiation protocol for obtaining pericytes / perivascular stromal cells (PSC) involved sequential growth factor treatment and is depicted in FIG. 1. The protocol is described in detail below.
[0132]NIH-registered ...
example 2
Morphological Characteristics and Scalable Growth of Pericyte-Like Cells of the Invention
[0140]The morphological characteristics of the cultured pericyte-like cells of the invention are illustrated in FIG. 2A. In comparison to primary pericytes (PL-PC and BV-PC, of placental and brain vessel origin, respectively), the 017-PC-A and 017-PC-M appeared as smaller spindle-shaped cells that grew to a high density. After about 18-20 passages, the 017-PC-A and 017-PC-M cells became bigger in size, indicative of hypertrophic senescence, while still exhibiting the same spindle-like shape.
[0141]Both 017-PC-A and 017-PC-M expanded well in their corresponding media, and have potential to reach commercial scale, as illustrated in FIG. 2B.
example 3
Phenotype / Marker Expression Analysis of HESC-Derived Pericyte-Like Cells of the Invention
[0142]017-PC-A and 017-PC-M cells as well as clonal lines E164 and CMO2 and control placental (PL-PC) and brain-vessel (BV-PC) primary pericytes were analyzed for expression of a number of surface markers. The markers tested were grouped as following: 1) pericyte: CD146, CD105, PDGFRβ and NG2; 2) mesenchymal: CD73 and CD90; 3) stemness: TRA-1-60, Oct-4, CD133 and podoplanin (PDPN); and 4) hematopoietic / endothelial: CD34, CD31, CD144 and CD45. The antibodies were obtained from commercial vendors as indicated. CD146, CD73, CD90, TRA-1-60, PDPN, CD34, CD31, CD133 and CD45 antibodies were from BioLegend Antibodies; Oct-4 antibody was from Chemicon; CD105 antibody was from BD Biosciences; PDGFRβ and NG2 antibodies were from R&D Systems; CD133 antibody was from Dako.
[0143]Marker expression was assessed using flow cytometry (Accurri C6 flow cytometer, BD Bioscience). The data was analyzed with FCS Expr...
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