PLURIPOTENT STEM CELLS, METHOD FOR PREPARATION THEREOF AND USES THEREOF

• US 20110312091A1
• Filed: 10/23/2009
• Published: 12/22/2011
• Est. Priority Date: 12/17/2008
• Status: Abandoned Application

• Current Assignee: Beijing Health & Biotech (H & B) Co., Ltd.
• Original Assignee: Beijing Health & Biotech (H & B) Co., Ltd.
• Inventors: Zhao, Baona, Han, Zhihai
• Application Number:
  US13/140,581
• Publication Number:
  US 20110312091A1
• Time in Patent Office: Days
• US Class Current: 435/368
• CPC Class Codes: A61K 2035/122 for inducing tolerance or supression of immune responses
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 39/06 Free radical scavengers or antioxidants
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696

Abstract

Human pluripotent stem cells which are isolated from cut human umbilical cord or placenta and characteristic of cell surface marker CD151⁺, OCT4⁺ and CD184⁻, can adhere to tissue culture plastic and have the potential to differentiate into three germ layers: endoderm, mesoderm and ectoderm. Methods of isolating, purifying and culturally expanding of a population of human pluripotent stem cells and uses for treating diseases caused by cell damage or cell aging, and as a kind of carrier cells in gene therapy are provided.

Claims

1. An isolated population of pluripotent stem cells derived from human umbilical cord or placenta which express CD151 and OCT4, at the same time lack expression of CD184.

2. The pluripotent stem cells of claim 1 wherein said pluripotent stem cells also express Sox-2, Nestin, CD90, CD29, CD13, CD166, CD105, CD44, CD73, CD49e, GD2 and HLA-I, and lack expression of CD34, CD31, CD45, CD133, CD106, CD11b, CD271, CD41, CD61, CD42b and HLA-II.

3. The pluripotent stem cells of claim 1 wherein said pluripotent stem cells can adhere to tissue culture plastic and have the potential to differentiate into endoderm, mesoderm and ectoderm, additionally, form none of teratoma after subcutaneous injection into nude mice.

4. The pluripotent stem cells of claim 3 wherein said endoderm, mesoderm and ectoderm are blood vessel, nerve, hepar, myocardium, bone, cartilage and adipose tissue.

9. The pluripotent stem cells of claim 1 wherein pluripotent stem cells are used as carrier cells in gene therapy.

10. The pluripotent stem cells of claim 1 wherein pluripotent stem cells are used for treating diseases caused by cell damage or cell aging.

11. The pluripotent stem cells of claim 1 wherein pluripotent stem cells are used for promoting differentiation of stem cells into lipoblast, osteoblast, chondroblast, myocardial cell, nerve cell, hepatocyte and stimulating the haematogenesis.

12. The pluripotent stem cells of claim 1 wherein pluripotent stem cells are used for treating diseases of immunologic abnormality.

13. The pluripotent stem cells of claim 1 wherein pluripotent stem cells are used for treating brain damage.

14. The pluripotent stem cells of claim 1 wherein pluripotent stem cells are used for treating graft-versus-host disease.

15. The pluripotent stem cells of claim 2 wherein said pluripotent stem cells can adhere to tissue culture plastic and have the potential to differentiate into endoderm, mesoderm and ectoderm, additionally, form none of teratoma after subcutaneous injection into nude mice.

5. A method of isolating, purifying and culturally expanding of a population of human pluripotent stem cells from cut human umbilical cord or placenta, a process comprising:

cutting and collecting human umbilical cord and or placenta tissues by aseptic processing;

mincing collected tissues into fragments and incubating the fragments with protease and then passing through a filter to obtain primary mononuclear cells;

seeding the mononuclear cells in culture flasks and maintaining in growth medium I or II and expanding cells through passage 4 or above; and

collecting the cells and cryopreserving in liquid nitrogen;

wherein the growth medium I is comprised of 50-70% DMED/F12 and 30-50% MCDB-201, supplemented with 2-10% serum, 10<sup>−

8</sup> mol/L dexamethasone, 10-50 mg/mL insulin-transferrin-selenium (ITS), 0.1-10 mmol/L glutamine, 1-100 ng/mL human epidermal growth factor (hEGF) and 1-100 ng/mL basic fibroblast growth factor (bFGF); and

the growth medium II is comprised of 50-70% DMED/F12 and 30-50% MCDB-201, supplemented with 0.1-5% (W/V) human serum albumin (HAS), 1-100 μ

g/mL linolenic acid, 1-100 μ

g/mL linoleic acid, 0.1-5% non-essential amino acid, 10<sup>−

8</sup> mol/L dexamethasone, 10-50 mg/mL insulin-transferrin-selenium (ITS), 0.1-10 mmol/L glutamine, 1-100 ng/mL human epidermal growth factor (hEGF) and 1-100 ng/mL basic fibroblast growth factor (bFGF).

6. The method of claim 5 wherein the protease is collagenase type I or trypsin.

7. A method of isolating, purifying and culturally expanding of a population of human pluripotent stem cells from cut human umbilical cord or placenta, a process comprising:

cutting and collecting human umbilical cord and/or placenta tissues by aseptic processing;

mincing collected tissues into fragments and incubating the fragments with protease and then passing through a filter to obtain primary mononuclear cells; and

selecting CD151+, OCT4+ and CD184⁻

cells from the mononuclear cells through Magnetic Activated Cell Sorting and cryopreserving in liquid nitrogen.

8. The method of claim 7 wherein the protease is collagenase type I or trypsin.