Multipotent stem cells and uses thereof

a multi-potent stem cell, stem cell technology, applied in the direction of skeletal/connective tissue cells, drug compositions, immunological disorders, etc., can solve the problems of critical shortage of stem cells, difficult to obtain sufficient human stem cells, and difficult to achieve embryonic-like effects

Inactive Publication Date: 2010-11-18
THE BRIGHAM & WOMEN S HOSPITAL INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]A purified population of stem cells that exist in the synovial fluid and blood is disclosed. These cells are embryonic in character and prior to culture do not present the surface markers generally associated with other adult stem cells, even after days in culture. These cells also express key embryonic transcription factors within a few days of isolation, in contrast to other adult stem cells, which require longer periods of culture before such expression. Further, these cell can differentiate into all three germ layers (mesoderm, ectoderm, and endoderm) and do not form teratoma bodies in vitro. This discovery allows for a non-controversial supply of easily attainable embryonic-like stem cells.

Problems solved by technology

Prior to the present invention, a basic problem existed, i.e., that obtaining sufficient quantities and populations of human stem cells which are capable of differentiating into most cell types was nearly impossible.
Stem cells are in critically short supply.
Obtaining sufficient numbers of human stem cells has been problematic for several reasons.
First, isolation of normally occurring populations of stem cells in adult tissues has been technically difficult and costly due, in part, to very limited quantity found in blood or tissue.
The isolation of the cells is generally laborious, involving the harvesting of cells or tissues from a patient or donor, culturing and / or propagating the cells in vitro.
Even in cell types that are replaced in adult organisms (e.g., epithelial cells and hematopoietic cells), it has been a significant challenge to readily and inexpensively obtain stem cells in significant quantities.
However, these hematopoietic stem cells are very rare in adults, accounting for approximately 0.01% of bone marrow cells.
Isolation of these cells based on surface proteins such as CD34 results in very low yields.
Schemes to fractionate human hematopoietic cells into lineage committed and non-committed progenitors are technically complicated and often do not permit the recovery of a sufficient number of cells to address multilineage differentiation (Berenson et al., 1991; Terstappen et al., 1991; Brandt et al., J. Clin. Invest. 82:1017-1027, 1988; Landsdorp et al., J. Exp. Med. 175:1501-1509, 1992; Baum et al., Proc. Natl. Acad. Sci.
A second reason that obtaining sufficient number of human stem cells has been problematic is that procurement of these cells from embryos or fetal tissue has raised religious, ethical, and legal concerns.
Many adult stem cell propagation protocols require such cells, which creates risks including infection, cell fusion, and / or contamination.
As such, adult stem cells are have often been very difficult to expand in culture (Reya et al., Nature 414:105-111, 2001; Tang et al.

Method used

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  • Multipotent stem cells and uses thereof
  • Multipotent stem cells and uses thereof
  • Multipotent stem cells and uses thereof

Examples

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example 1

Purifying and Enriching Stem Cells from Synovial Fluid

[0195]In a first step, freshly isolated synovial fluid was obtained from an osteoarthritic (OA) patient. Synovial fluid mononuclear cells were thus derived from joint aspirates. Joint aspirates can be obtained by standard methods well known to those of skill in the art.

[0196]Harvesting SF Stem Cells (Heterogenous Population).

[0197]Synovial fluid (SF) from OA patients was harvested, diluted in serum-free medium (AIM-V, GIBCO) or MCDB, MEM, IMDM, RPMI media, and spun at 200 g for 15 minutes at room temperature (RT). The pelleted population was then resuspended in AIM-V up to the original SF volume. The mononuclear cell number from SF ranged between 500,000 to 5 million heterologous mononuclear cells.

[0198]Harvesting SF Stem Cells (Homogenous Population)

[0199]The pelleted population was then resuspended in AIM-V, serum free medium developed for the ex vivo expansion of human lymphocytes, up to the original SF volume, counted, washed...

example 2

Characterization and Expansion of Enriched Stem Cells

[0203]Both populations of separated cells (i.e., from the buffy layer or the pellet) were incubated with anti-MHC class I, anti-CD66b, and anti-glycophorin b, immunomagnetic beads as described above. Samples were first assessed for the expression of MHC class I, MHC class II, CD44, CD45, CD13, CD34, CD49c, CD66b, CD73, CD105 and CD90 cell surface markers and then permeabilized to determine the presence of Oct-4, Nanog, Sox-2, Rex-1, GDF-3, and Stella intracellularly. FIGS. 1a-1e are dot plots of the enriched cells, sorted into 3 groups (e.g., Group-A, -B, and -C) and analyzed for Oct-4, Rex-1, Runx2, Sox-9, Nanog, Class I, CD44, and CD45 expression. Six to thirty percent of total mononuclear population, i.e., groups A and B, expressed Oct-4, Nanog, Sox-9, and Rex-1 while group C expressed the aforementioned transcription factors at high levels, with the exception of Oct-4. The majority of the cells were negative for the above cell...

example 3

Propagation of Synovial Fluid Stem Cells

[0204]Heterogenous or homogenous populations of SF stem cells not incubated with immunomagnetic beads were plated onto culture dishes coated with about 7-10 ng / ml serum fibronectin or other appropriate matrix coating. Cells were maintained in Dulbecco Minimal Essential Medium (DMEM) or 40-60% (e.g., 60%) Low Glucose DMEM and 40-60% (e.g., 40%) MCDB-201 medium supplemented with 1-50 ng / ml (e.g., about 5-15 ng / ml or 10 ng / ml) platelet-derived growth factor-BB (PDGF-BB), 1-50 ng / ml (e.g., about 5-15 ng / ml or 10 ng / ml) epidermal growth factor (EGF), 1-50 ng / ml (e.g., about 5-15 ng / ml or 10 ng / ml) insulin-like growth factor (IGF), or 100-10,000 IU (e.g., about 1,000) LIF, with 10−11 to 10−8 M (e.g., 0.01 nM) dexamethasone or other appropriate steroid(s), 2-10 μg / ml (e.g., 4.7 ng / ml) linoleic acid, and 0.05-0.15 μm (e.g., 0.1 μm) ascorbic acid. The culture medium may further include 10-50 ng / ml (e.g., 10 ng / ml) insulin, 0-10 ng / ml (e.g., 5.5 ng / ml) ...

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Abstract

The invention provides a quiescent stem cell having the capacity to differentiate into ectoderm, mesoderm and endoderm, and which does not express cell surface markers including MHC class I, MHC class II, CD44, CD45, CD13, CD34, CD49c, CD73, CD105 and CD90. The invention further provides a proliferative stem cell, which expresses genes including Oct-4, Nanog, Sox2, GDF3, P16INK4, BMI, Notch, HDAC4, TERT, Rex-1 and TWIST but does not express cell surface markers including MHC class I, MHC class II, CD44, CD45, CD13, CD34, CD49c, CD73, CD105 and CD90. The cells of the invention can be isolated from adult mammals, have embryonic cell characteristics, and can form embryoid bodies. Methods for obtaining the stem cells, as well as methods of treating diseases and differentiated the stem cells, are also provided.

Description

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH [0001]This work was supported by NIAMS grant AR050243. The government may have certain rights in this invention.BACKGROUND OF THE INVENTION[0002]A stem cell is commonly defined as a cell that (i) is capable of renewing itself; and (ii) can give rise to more than one type of cell through asymmetric cell division (Watt et al., Science, 284:1427-1430, 2000). Stem cells typically give rise to a type of multipotent cell called a progenitor cell; progenitor cells, in turn, proliferate and differentiate into lineage-committed cells that populate the body.[0003]Pluripotent stem cells are thought to have the potential to differentiate into almost any cell type, while multipotent stem cells are believed to have the potential to differentiate into many cell types (Robertson, Meth. Cell Biol. 75:173, 1997; Pedersen, Reprod. Fertil. Dev. 6:543-552, 1994).[0004]Stem cells exist in many tissues of embryos and adult mammals....

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/12C12N5/071C12N5/10A61P37/02A61P9/00A61P17/02C12N5/074C12N5/0775
CPCC12N2500/25C12N5/0662C12N2500/38C12N2501/105C12N2501/11C12N2501/135C12N2501/155C12N2501/2303C12N2501/2306C12N2501/235C12N2501/39C12N2501/41A61K35/12C12N5/0668C12N2500/36A61P17/02A61P37/02A61P9/00A61K35/14A61K35/24C12N5/0639C12N5/0647C12N5/0653C12N5/0654C12N5/0655C12N5/0657C12N5/0658C12N5/0661C12N5/067C12N5/0676C12N2501/125C12N2501/22C12N2501/26C12N2501/40C12N2501/999
Inventor CRAWFORD, KEITH W.SOUTHGATE, CHRISTOPHER
Owner THE BRIGHAM & WOMEN S HOSPITAL INC
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