Methods, kits, and compositions for stem cell self-renewal

a technology of stem cell self-renewal and kits, applied in the field of hematopoietic stem cell population expansion, can solve the problems of pten in stem cell stem cell and tumorigenesis, recurrence of tumors heretofore not understood, and a risk of over-activation and expansion of stem cells,

Inactive Publication Date: 2010-04-22
STOWERS INST FOR MEDICAL RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0018]Thus, one embodiment of the invention is an ex vivo method for expanding the number of hematopoietic stem cells (HSC) in a population of mononuclear cells (MNC). This method comprises culturing the population of MNCs comprising at least one HSC in an HSC expansion media for a period of time sufficient to expand the number of HSCs in the MNC population, wherein the expanded HSCs are functional with long term, multi-lineage, repopulating potential.
[0021]Yet another embodiment of the invention is an ex vivo method for expanding the number of cells capable of supporting multi-lineage repopulation in a population of mononuclear cells (MNC). This method comprises culturing the population of MNCs comprising at least one hematopoietic stem cell (HSC) and at least one hematopoietic progenitor cell in an HSC expansion media for a period of time sufficient to expand the number of cells capable of supporting multi-lineage repopulation in the MNC population.
[0022]Another embodiment of the invention is a method for expanding a population of stem cells obtained from a tissue selected from the group consisting of peripheral blood, cord blood, and bone marrow. This method comprises modulating a PTEN pathway and a Wnt pathway in the population of stem cells to expand the number of stem cells.

Problems solved by technology

Over-activation and expansion of stem cells risks both eventual depletion of the stem cell population and a predisposition to tumorigenesis.
But, the role of PTEN in stem cells and tumorigenesis and the recurrence of tumors heretofore has been not understood.
FoxO factors induce expression of p27, which can bind to cyclin E / cdk2 complexes and inhibit their activity, resulting in a block in cellular proliferation.

Method used

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  • Methods, kits, and compositions for stem cell self-renewal
  • Methods, kits, and compositions for stem cell self-renewal
  • Methods, kits, and compositions for stem cell self-renewal

Examples

Experimental program
Comparison scheme
Effect test

example 2

In Vitro Culture of Control and Mutant LSK Cells

Cell Culture

[0217]LSK or LSK Flk2− cells were sorted into 96-well U-bottom tissue culture plates at 100 cells / well with 200 μl media / well. Cells were incubated at 37° C., 5% O2, 5% CO2 (balance N2) for the indicated number of days. One-half total volume of media (see Table 1, below for the base media) was carefully pipetted from the top and replaced with fresh media every other day.

TABLE 1Base MediaComponentsSourceStemSpan Media: (Iscove's-modified Dulbecco'sStem Cellmedium (IMDM) supplemented with 1% bovineTechnologies;serum albumin, 10 μg ml−1 recombinant humanCat. No. 09600insulin, 200 μg ml−1 iron-saturated transferrin,0.1 mM 2-mercaptoethanol and 2 mM glutamine.)10 μg / ml HeparinSigma, Cat.No. H-31490.5X Penicillin / StreptomycinSigma, Cat.No. P433310 ng / ml recombinant mouse (rm) Stem CellBiovision, Cat.FactorNo. 4328-1020 ng / ml rm-ThrombopoietinCell Sciences,Inc, Cat. No.CRT401B

Double Mutant HSCs Expand Dramatically In Vitro and In ...

example 3

Transplantation Analysis of Pten and Pten:Ctnnb1 LSK Cells After 5 Weeks of Culture

[0220]For the following experiments, cells were cultured in the same manner as described in Example 2. As in Example 2, the base media of Table 1 was supplemented with 20 ng / ml rm-IGF-2 (R&D Systems, 792-MG) and 10 ng / ml recombinant human FGF-1 (Affinity BioReagents, ORP16010).

[0221]While Pten and especially Pten:Ctnnb1 cultures exhibited significant expansion in LSK cells, whether these cells were functional in vivo was determined.

[0222]At 5 weeks culture, Pten and Pten:Ctnnb1 LSK cultures were re-sorted and 1000 LSK cells (CD45.2+) from each were transplanted into lethally irradiated (10Gy) CD45.1+ recipient mice along with 2×105 congenic whole bone marrow competitor cells. Because wild-type cells did not survive 5 weeks culture, 1000 fresh wild-type LSK cells were also transplanted as a separate control group. Peripheral blood analysis at 4 weeks post-transplantation revealed robust repopulation in...

example 4

Differentiation Block and Dominant Phenotype of Pten:Ctnnb1 Mutant HSCs

[0225]Initially, primary (non-transplanted) animals were used for phenotypic analysis. These mice eventually exhibited severe non-hematopoietic defects, including reduction of the marrow cavity and splenic fibrosis resulting in disruption of splenic niches (FIG. 16). Consequently, LT-HSC transplantations were used to verify Scl-Cre specificity (FIG. 10). Comparing these transplant groups with the initial data from primary mutants revealed an essentially identical phenotypic manifestation of defects between transplant and non-transplant groups, demonstrating that non-hematopoietic effects are due to interaction between the hematopoietic system and stroma rather than from defects arising from the stroma (FIG. 17 and data not shown).

[0226]The health of double mutants typically declined by 9 wpi (see below). LSK cells and early progenitors from control, single, and double mutant bone marrow and spleen at 9-10 wpi wer...

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Abstract

The present invention relates to methods and kits for expanding a stem cell population. More particularly, the invention relates, inter alia, to methods, kits, and compositions for expanding a stem cell population, particularly a hematopoietic stem cell population.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation-in-part of International Application Serial No. PCT / US2008 / 005230, filed Apr. 23, 2008, which claims benefit to U.S. Provisional Patent Application Ser. No. 60 / 926,065, filed Apr. 23, 2007 and U.S. Provisional Patent Application Ser. No. 61 / 066,693, filed Feb. 22, 2008. The entire contents of the above-mentioned applications are hereby incorporated by reference as if recited in full herein.FIELD OF THE INVENTION[0002]The present invention relates to methods, kits, and compositions for expanding a stem cell population, particularly an hematopoietic stem cell population.BACKGROUND OF THE INVENTION[0003]Hematopoietic stem cells (HSCs) are clonogenic cells, which possess the properties of both self-renewal (expansion) and multilineage potential giving rise to all types of mature blood cells. HSCs are responsible for hematopoiesis and undergo proliferation and differentiation to produce mature blood cells of ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/02C12N5/00C12N5/0789
CPCC12N5/0647C12N2501/70C12N2501/415C12N2501/40A61P3/00A61P7/00A61P7/06A61P35/00A61P35/02A61P43/00A61K35/28C12N2502/11
Inventor PERRY, JOHN M.LI, LINHENGGRINDLEY, JUSTIN C.
Owner STOWERS INST FOR MEDICAL RES
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