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Generation of clonal mesenchymal progenitors and mesenchymal stem cell lines under serum-free conditions

Inactive Publication Date: 2010-10-14
WISCONSIN ALUMNI RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0032]It is still another advantage of the invention that MSCs obtained by the claimed methods are of mesodermal origin and can be derived from APLNR+ cells enriched in lateral plate mesoderm cells.

Problems solved by technology

The art is limited by an inability to isolate sufficient MSCs for subsequent differentiation and use.
Where suitable donors are available, the invasive procedures required to isolate even a limited number of cells present risks to donors.
It also remains difficult to maintain isolated MSCs in long-term culture and to maintain such cultures free of bacterial or viral contamination.

Method used

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  • Generation of clonal mesenchymal progenitors and mesenchymal stem cell lines under serum-free conditions
  • Generation of clonal mesenchymal progenitors and mesenchymal stem cell lines under serum-free conditions
  • Generation of clonal mesenchymal progenitors and mesenchymal stem cell lines under serum-free conditions

Examples

Experimental program
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Effect test

example 1

[0061]Generation of MSCs from Pluripotent Stem Cells Under Serum-Free Conditions.

[0062]hESCs (H1; WiCell; Madison, Wis.) were maintained on irradiated mouse embryonic fibroblasts in a serum-free medium, such as DMEM / F12 medium supplemented with 20% Knockout™ serum replacer, 2 mM L-glutamine, 1×(100 μM) non-essential amino acids, 100 μM 2-mercaptoethanol and 4 ng / ml bFGF (all from Gibco-Invitrogen; Carlsbad, Calif.). See Amit M, et al., “Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture,” Dev. Biol. 227:271-278 (2000), incorporated herein by reference as if set forth in its entirety. Mouse OP9 bone marrow stromal cells (kindly provided by Dr. Toru Nakano and available from ATCC, catalog # CRL-2749) were maintained by four-day subculture on gelatin-coated dishes in alpha MEM medium (Gibco-Invitrogen) with 20% fetal calf serum (FCS; HyClone; Logan, Utah).

[0063]The hESCs were induced to differentiate by co...

example 2

[0076]In vitro generation and characterization of mesangioblasts.

[0077]To isolate and characterize a population of mesodermal progenitors that can give rise to cells of the mesodermal lineage with hematopoietic, endothelial, and mesenchymal stem cell potentials, H1 hES cells were co-cultured with OP9 cells, as described in Example 1. After two or three days of co-culture, when genes representative of primitive streak population (mesendoderm) (MIXL1, T, EOMES) were expressed, the hESC-derived cells depleted of OP9 cells using anti-mouse CD29 antibody were plated in semisolid, serum-free medium, essentially as described in Example 1, with 20 ng / ml bFGF (PeproTech; Rocky Hill, N.J.). The number of colony-forming cells (CFCs) was calculated per 1000 plated H1-derived TRA-1-85+ cells.

[0078]After 2-3 days in semisolid medium, the cells formed tightly packed structures (cores). Cores derived from hESCs that were differentiated in co-culture with OP9 cells for 2 days further grew into spher...

example 3

[0083]Generating and isolating a population of cells substantially enriched in lateral plate / extraembryonic mesoderm cells.

[0084]Genetic profiling of H1 hESCs differentiated in OP9 cocultures demonstrated selective commitment toward mesodermal and endodermal lineages with no detectable ectoderm (tropho-, neuro-, or surface ectoderm) (FIG. 2). The cells became committed to mesendoderm by day 2 of culture, when synchronous expression of primitive streak genes (MIXL1, T, and EOMES) was detected. At subsequent days of culture, mesoderm- and endoderm-specific genes and, eventually, endoderm- and mesoderm derivative-specific genes were expressed. Of the mesodermal genes, those characteristic of the lateral plate / extraembryonic mesodermal subset (FOXF1, HAND1, NKX2-5, GATA2) were expressed consistently, while expression of genes of the axial (CHRD, SHH), paraxial (MEOX1, TCF15), or intermediate (PAX2, PAX8) subsets was not consistent. Apelin receptor (APLNR) expression is strongly induced ...

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Abstract

Methods for obtaining multipotent Apelin receptor-positive lateral plate mesoderm cells, mesenchymal stem cells, and mesangioblasts under serum-free conditions are disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation-in-part application of U.S. patent application Ser. No. 12 / 554,696, which is a continuation application of U.S. patent application Ser. No. 12 / 024,770, which claims the benefit of U.S. Provisional Patent Application No. 60 / 974,980, filed Sep. 25, 2007; and U.S. Provisional Patent Application No. 60 / 989,058, filed Nov. 19, 2007, each of which is incorporated herein by reference as if set forth in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This invention was made with United States government support awarded by the following agency: NIH RR052085 and NIH HD044067. The United States government has certain rights in this invention.BACKGROUND[0003]The invention relates generally to clonal primate mesenchymal progenitors and to mesenchymal stem cell (MSC) lines and methods for identifying and generating such cells, and more particularly to methods for generating clonal mes...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0647C12N2502/1394C12N5/069C12N5/0692C12N2500/38C12N2500/44C12N2500/99C12N2501/115C12N2501/125C12N2501/135C12N2501/145C12N2501/155C12N2501/165C12N2501/23C12N2506/02C12N2506/45C12N2533/54C12N2533/70C12N2533/78C12N5/0662C12N2500/90
Inventor VODYANYK, MAKSYM A.SLUKVIN, IGOR I.
Owner WISCONSIN ALUMNI RES FOUND
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