Title of Invention	Process of isolating Mesenchymal Stem Cells
Abstract	The present invention provides a process of isolation, proliferation and/or maintenance of mesenchymal stem cells (MSCs).The invention further provides a culture medium for proliferation and/or maintenance of human mesenchymal stem cells in xeno-free conditions.The culture medium provides provided in the present invention proliferates and/or maintains mesenchymal stem cell expansion while maintaining a multipotent phenotype

Title of Invention

Process of isolating Mesenchymal Stem Cells

Abstract

The present invention provides a process of isolation, proliferation and/or maintenance of mesenchymal stem cells (MSCs).The invention further provides a culture medium for proliferation and/or maintenance of human mesenchymal stem cells in xeno-free conditions.The culture medium provides provided in the present invention proliferates and/or maintains mesenchymal stem cell expansion while maintaining a multipotent phenotype

Full Text	I/We Claim: 1. A process of isolation of mesenchymal stem cells (MSC) from a sample, said process comprising; a) treating said sample with an antibody against an antigen, wherein said antigen is selected from a group consisting of CD44, CD50, CD54, CD73, CD90, CD 105, CD 106, CD 117, oct 3/4, Actin filament associated protein (AFAP), Frizzled7 (FZD7), DickkopG (DKK3), Protein tyrosine phosphatase receptor F (PTPRF), RAB3B, and Stro-1; b) obtaining cells expressing said antigen; wherein said cells are mesenchymal stem cells; and c) culturing said mesenchymal stem cells in a growth medium selected from the group consisting of i) a culture medium comprising a basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; 5-10% fetal bovine serum (FBS), growth factors, 200 mM glutamax and antibiotics; ii) a culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors, human serum, 200 mM glutamax and antibiotics; iii) a culture medium comprising basal medium selected from the group consisting of KO- DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors, human plasma, heparin, 200 mM glutamax and antibiotics; and iv) a culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors, human plasma or human serum or a combination thereof, heparin, 200 mM glutamax and antibiotics. 2. The process as claimed in claim 1, wherein said process further comprises maintaining said mesenchymal stem cells after culturing as in step (c) of claim 1 in a maintenance medium selected from the group consisting of i) a culture medium comprising a basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; 5-10% fetal bovine serum (FBS), growth factors, 200 mM glutamax and antibiotics; ii) a culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors, human serum, 200 mM glutamax and antibiotics; iii) a culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors, human plasma, heparin, 200 mM glutamax and antibiotics; and iv) a culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors, human plasma or human serunj or a combination thereof, heparin, 200 mM glutamax and antibiotics; 3. The process as claimed in claim 1 or 2, wherein said sample is selected from the group consisting of bone marrow, corneal epithelial tissue, adipose tissue, umbilical cord, placenta, dental ligament and dental pulp. 4. The process as claimed in claim 1 or 2, wherein said sample is bone marrow. 5. The process as claimed in claim 1 or 2, wherein said sample is corneal epithelial tissue. 6. The process as claimed in claim 1 or 2, wherein said sample is adipose tissue. 7. The process as claimed in claim 1 or 2, wherein said sample is umbilical cord. 8. The process as claimed in claim 1 or 2, wherein said sample is placenta. 9. The process as claimed in claim 1 or 2, wherein said sample is dental ligament. 10. The process as claimed in claim 1 or 2, wherein said sample is dental pulp. 11. The process as claimed in claim 1 or 2, wherein said sample is obtained from human or murine origin. 12. The process as claimed in claim 1 or 2, wherein said growth medium comprises a basal medium KO-DMEM, 5-10% fetal bovine serum (FBS), growth factors, 200 mM glutamax and antibiotics. 13. The process as claimed in claim 1 or 2, wherein said growth factors are selected from the group consisting of platelet derived growth factor (PDGF) 5 to 100 r\|g/ml, transforming growth factor-beta (TGF-beta) 5 to 50r)g /ml, keratinocyte growth factor (KGF) 4 to 100r\|g/ml, basic Fibroblast growth factor (bFGF) 4 to 80 r)g/ml, Epidermal growth factor (EGF) 5 to 50 r\|g/ml and Insulin-like growth factor-I (IGF- I) 5 to 50r\|g /ml. 14. The process as claimed in claim 1 or 2, wherein concentration of PDGF 20r\|g/ml. 15. The process as claimed in claim 1 or 2, wherein concentration of TGF is 20rig /ml. 16. The process as claimed in claim 1 or 2, wherein concentration of said KGF is a 20r\|g /ml. • 17. The process as claimed in claim 1 or 2, wherein concentration of bFGF is 10 rjg/ml. 18. The process as claimed in claim 1 or 2, wherein concentration of EGF is 15 rig/ml. 19. The process as claimed in claim 1 or 2, wherein concentration of IGF-I is 20r(g /ml. 20. The process as claimed in claim 1 or 2, wherein concentration of human serum is in the range of 0.5 to 1% (w/w). 21. The process as claimed in claim 1 or 2, wherein concentration of human plasma is in the range of 1 to 20 %(w/w). 22. The process as claimed in claim 1 or 2, wherein concentration of glutamax is 200 mM. 23. The process as claimed in claim 1 or 2, wherein concentration of heparin is in the range of 1000 tolO, 000 U/ml. 24. The process as claimed in claim 1 or 2, wherein said MSCs are negative for a marker selected from the group consisting of CD3, CD4, CD8, CD10, CD13, CD14, CD19, CD29, CD31, CD34, CD36, CD38, CD45, CD62E, CD66b, CD133, HLA I and II, HLA-DR, glycophorin-A, Mucl8, cKit, Tie/Tek, microglobulin, oct 4, Nanog, Rex- 1, TDGF, TERT, SOX-2 and beta actin control. 25. The process as claimed in claim 1 or 2, wherein said mesenchymal stem cells retain the functional characteristics without any abnormalities upto more than 30 passages. 26. The process as claimed in claim 1 or 2, wherein said mesenchymal stem cells retain the fiinctional characteristics without any abnormalities upto 30 passages. 27. The process as claimed in claim 1 or 2, wherein said mesenchymal stem cells are capable of differentiation into cells selected from a group consisting of cardiac cells, neuronal cells, hepatocytes, pancreatic beta cells, osteoblasts, chondrocytes and adipocytes. 28. A cell culture medium for proliferation and/or maintenance of mesenchymal stem cells (MSCs), wherein said culture medium comprising KO-DMEM; growth factors selected from the group consisting of 5 to lOOrjg/ml platelet derived growth factor (PDGF), 5 to 50r\|g /ml transforming growth factor-beta (TGF-beta), 4 to 100r)g/ml keratinocyte growth factor (KGF), 4 to 80 r^g/ml basic Fibroblast growth factor (bFGF), 5 to 50 ng/ml Epidermal growth factor (EGF) and 5 to 50rig /ml Insulin-like growth factor-I (IGF-I); 5 to 10% (w/w) fetal bovine serum (FBS); 200 mM glutamax; and antibiotics. 29. A cell culture medium for proliferation and/or maintenance of mesenchymal stem cells (MSCs), wherein said culture medium comprising KO-DMEM; growth factors selected from the group consisting of 5 to 100rig/ml platelet derived growth factor (PDGF), 5 to 50r\|g /ml transforming growth factor-beta (TGF-beta), 4 to 100Tig/ml keratinocyte growth factor (KGF), 4 to 80 rig/ml basic Fibroblast growth factor (bFGF), 5 to 50 r\|g/ml Epidermal growth factor (EGF) and 5 to 50r\|g /ml Insulin-like growth factor-I (IGF-I);human plasma or human serum or a combination thereof; 200 mM glutamax; and antibiotics. 30. A cell culture medium for proliferation and/or maintenance of mesenchymal stem cells (MSCs), wherein said culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors selected from the group consisting of 5 to 100 rig/ml platelet derived growth factor (PDGF), 5 to 50Tig/ml transforming growth factor-beta (TGF-beta), 4 to I00r\|g/mi keratinocyte growth factor (KGF), 4 to 80 rig/ml basic Fibroblast growth factor (bFGF), 5 to 50 rig/ml Epidermal growth factor (EGF) and 5 to 50rig/ml Insulin-like growth factor-I (IGF-I); 0.5 to 1% human serum; 200 mM glutamax; and antibiotics. 31. A cell culture medium for proliferation and/or maintenance of mesenchymal stem cells (MSCs), wherein said culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors selected from the group consisting of 5 to 100 rig/ml platelet derived growth factor (PDGF), 5 to 50rig/ml transforming growth factor-beta (TGF-beta), 4 to 100r)g/ml keratinocyte growth factor (KGF), 4 to 80 r\|g/m! basic Fibroblast growth factor (bFGF), 5 to 50 r\|g/ml Epidermal growth factor (EGF) and 5 to 50rig/ml Insulin-like growth factor-I (IGF-I); 1 to 20 % human plasma; 200 mM glutamax; 1000 to 10,000 U/ml heparin and antibiotics. 32. A cell culture medium for proliferation and/or maintenance of mesenchymal stem cells (MSCs), wherein said culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors selected from the group consisting of 5 to 100 rig/ml platelet derived growth factor (PDGF), 5 to 50rig/ml transforming growth factor-beta (TGF-beta), 4 to 100rig/ml keratinocyte growth factor (KGF), 4 to 80 rig/ml basic Fibroblast growth factor (bFGF), 5 to 50 rig/ml Epidermal growth factor (EGF) and 5 to 50iig/ml Insulin-like growth factor-I (IGF-I); 0.5 to 1% human serum or 1 to 20 % human plasma; 200 mM glutamax; 1000 to 10,000 U/ml heparin and antibiotics. ^

Full Text

I/We Claim:
1. A process of isolation of mesenchymal stem cells (MSC) from a sample, said
process comprising;
a) treating said sample with an antibody against an antigen, wherein said antigen is selected from a group consisting of CD44, CD50, CD54, CD73, CD90, CD 105, CD 106, CD 117, oct 3/4, Actin filament associated protein (AFAP), Frizzled7 (FZD7), DickkopG (DKK3), Protein tyrosine phosphatase receptor F (PTPRF), RAB3B, and Stro-1;
b) obtaining cells expressing said antigen; wherein said cells are mesenchymal stem cells; and
c) culturing said mesenchymal stem cells in a growth medium selected from the group consisting of i) a culture medium comprising a basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; 5-10% fetal bovine serum (FBS), growth factors, 200 mM glutamax and antibiotics; ii) a culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors, human serum, 200 mM glutamax and antibiotics; iii) a culture medium comprising basal medium selected from the group consisting of KO- DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors, human plasma, heparin, 200 mM glutamax and antibiotics; and iv) a culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors, human plasma or human serum or a combination thereof, heparin, 200 mM glutamax and antibiotics.
2. The process as claimed in claim 1, wherein said process further comprises maintaining said mesenchymal stem cells after culturing as in step (c) of claim 1 in a maintenance medium selected from the group consisting of i) a culture medium comprising a basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; 5-10% fetal bovine serum (FBS), growth factors, 200 mM glutamax and antibiotics; ii) a culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors, human serum, 200 mM glutamax and antibiotics; iii) a culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors, human plasma, heparin, 200 mM glutamax and antibiotics; and iv) a culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors, human plasma or human serunj or a combination thereof, heparin, 200 mM glutamax and antibiotics;
3. The process as claimed in claim 1 or 2, wherein said sample is selected from the group consisting of bone marrow, corneal epithelial tissue, adipose tissue, umbilical cord, placenta, dental ligament and dental pulp.
4. The process as claimed in claim 1 or 2, wherein said sample is bone marrow.
5. The process as claimed in claim 1 or 2, wherein said sample is corneal epithelial tissue.
6. The process as claimed in claim 1 or 2, wherein said sample is adipose tissue.
7. The process as claimed in claim 1 or 2, wherein said sample is umbilical cord.
8. The process as claimed in claim 1 or 2, wherein said sample is placenta.
9. The process as claimed in claim 1 or 2, wherein said sample is dental ligament.
10. The process as claimed in claim 1 or 2, wherein said sample is dental pulp.
11. The process as claimed in claim 1 or 2, wherein said sample is obtained from human or murine origin.
12. The process as claimed in claim 1 or 2, wherein said growth medium comprises a basal medium KO-DMEM, 5-10% fetal bovine serum (FBS), growth factors, 200 mM glutamax and antibiotics.
13. The process as claimed in claim 1 or 2, wherein said growth factors are selected from the group consisting of platelet derived growth factor (PDGF) 5 to 100 r|g/ml, transforming growth factor-beta (TGF-beta) 5 to 50r)g /ml, keratinocyte growth factor (KGF) 4 to 100r|g/ml, basic Fibroblast growth factor (bFGF) 4 to 80 r)g/ml, Epidermal growth factor (EGF) 5 to 50 r|g/ml and Insulin-like growth factor-I (IGF- I) 5 to 50r|g /ml.
14. The process as claimed in claim 1 or 2, wherein concentration of PDGF 20r|g/ml.
15. The process as claimed in claim 1 or 2, wherein concentration of TGF is 20rig /ml.
16. The process as claimed in claim 1 or 2, wherein concentration of said KGF is a 20r|g /ml. •
17. The process as claimed in claim 1 or 2, wherein concentration of bFGF is 10 rjg/ml.
18. The process as claimed in claim 1 or 2, wherein concentration of EGF is 15 rig/ml.
19. The process as claimed in claim 1 or 2, wherein concentration of IGF-I is 20r(g /ml.
20. The process as claimed in claim 1 or 2, wherein concentration of human serum is in the range of 0.5 to 1% (w/w).
21. The process as claimed in claim 1 or 2, wherein concentration of human plasma is in the range of 1 to 20 %(w/w).
22. The process as claimed in claim 1 or 2, wherein concentration of glutamax is 200 mM.
23. The process as claimed in claim 1 or 2, wherein concentration of heparin is in the range of 1000 tolO, 000 U/ml.
24. The process as claimed in claim 1 or 2, wherein said MSCs are negative for a marker selected from the group consisting of CD3, CD4, CD8, CD10, CD13, CD14, CD19, CD29, CD31, CD34, CD36, CD38, CD45, CD62E, CD66b, CD133, HLA I and II, HLA-DR, glycophorin-A, Mucl8, cKit, Tie/Tek, microglobulin, oct 4, Nanog, Rex- 1, TDGF, TERT, SOX-2 and beta actin control.
25. The process as claimed in claim 1 or 2, wherein said mesenchymal stem cells retain the functional characteristics without any abnormalities upto more than 30 passages.
26. The process as claimed in claim 1 or 2, wherein said mesenchymal stem cells retain the fiinctional characteristics without any abnormalities upto 30 passages.
27. The process as claimed in claim 1 or 2, wherein said mesenchymal stem cells are capable of differentiation into cells selected from a group consisting of cardiac cells, neuronal cells, hepatocytes, pancreatic beta cells, osteoblasts, chondrocytes and adipocytes.
28. A cell culture medium for proliferation and/or maintenance of mesenchymal stem cells (MSCs), wherein said culture medium comprising KO-DMEM; growth factors selected from the group consisting of 5 to lOOrjg/ml platelet derived growth factor (PDGF), 5 to 50r|g /ml transforming growth factor-beta (TGF-beta), 4 to 100r)g/ml keratinocyte growth factor (KGF), 4 to 80 r^g/ml basic Fibroblast growth factor (bFGF), 5 to 50 ng/ml Epidermal growth factor (EGF) and 5 to 50rig /ml Insulin-like growth factor-I (IGF-I); 5 to 10% (w/w) fetal bovine serum (FBS); 200 mM glutamax; and antibiotics.
29. A cell culture medium for proliferation and/or maintenance of mesenchymal stem cells (MSCs), wherein said culture medium comprising KO-DMEM; growth factors selected from the group consisting of 5 to 100rig/ml platelet derived growth factor (PDGF), 5 to 50r|g /ml transforming growth factor-beta (TGF-beta), 4 to 100Tig/ml keratinocyte growth factor (KGF), 4 to 80 rig/ml basic Fibroblast growth factor (bFGF), 5 to 50 r|g/ml Epidermal growth factor (EGF) and 5 to 50r|g /ml Insulin-like growth factor-I (IGF-I);human plasma or human serum or a combination thereof; 200 mM glutamax; and antibiotics.
30. A cell culture medium for proliferation and/or maintenance of mesenchymal stem cells (MSCs), wherein said culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors selected from the group consisting of 5 to 100 rig/ml platelet derived growth factor (PDGF), 5 to 50Tig/ml transforming growth factor-beta (TGF-beta), 4 to I00r|g/mi keratinocyte growth factor (KGF), 4 to 80 rig/ml basic Fibroblast growth factor (bFGF), 5 to 50 rig/ml Epidermal growth factor (EGF) and 5 to 50rig/ml Insulin-like growth factor-I (IGF-I); 0.5 to 1% human serum; 200 mM glutamax; and antibiotics.
31. A cell culture medium for proliferation and/or maintenance of mesenchymal stem cells (MSCs), wherein said culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors selected from the group consisting of 5 to 100 rig/ml platelet derived growth factor (PDGF), 5 to 50rig/ml transforming growth factor-beta (TGF-beta), 4 to 100r)g/ml keratinocyte growth factor (KGF), 4 to 80 r|g/m! basic Fibroblast growth factor (bFGF), 5 to 50 r|g/ml Epidermal growth factor (EGF) and 5 to 50rig/ml Insulin-like growth factor-I (IGF-I); 1 to 20 % human plasma; 200 mM glutamax; 1000 to 10,000 U/ml heparin and antibiotics.
32. A cell culture medium for proliferation and/or maintenance of mesenchymal stem cells (MSCs), wherein said culture medium comprising basal medium selected from the group consisting of KO-DMEM, DMEM-LG and DMEM-F12 (1:1) or a combination thereof; growth factors selected from the group consisting of 5 to 100 rig/ml platelet derived growth factor (PDGF), 5 to 50rig/ml transforming growth factor-beta (TGF-beta), 4 to 100rig/ml keratinocyte growth factor (KGF), 4 to 80 rig/ml basic Fibroblast growth factor (bFGF), 5 to 50 rig/ml Epidermal growth factor (EGF) and 5 to 50iig/ml Insulin-like growth factor-I (IGF-I); 0.5 to 1% human serum or 1 to 20 % human plasma; 200 mM glutamax; 1000 to 10,000 U/ml heparin and antibiotics. ^

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Patent Number

279948

Indian Patent Application Number

861/CHE/2007

PG Journal Number

06/2017

Publication Date

10-Feb-2017

Grant Date

06-Feb-2017

Date of Filing

23-Apr-2007

Name of Patentee

STEMPEUTIC RESEARCH PRIVATE LIMITED

Applicant Address

AKSHAY TECH PARK #72 & 73 2ND FLOOR EPIP ZONE PHASE I-AREA WHITEFIELD BANGALORE 560066

Inventors:

#	Inventor's Name	Inventor's Address
1	SATISH MAHADEORAO TOTEY	9TH FLOOR, MANIPAL HOSPITAL, AIRPORT ROAD, BANGALORE-560017
2	UDAY KUMAR KOLKUNDKAR	9TH FLOOR, MANIPAL HOSPITAL, AIRPORT ROAD, BANGALORE-560017
3	RAKHI PAL	9TH FLOOR, MANIPAL HOSPITAL, AIRPORT ROAD, BANGALORE-560017
4	MADHIRI HANWATE	9TH FLOOR, MANIPAL HOSPITAL, AIRPORT ROAD, BANGALORE-560017

PCT International Classification Number

C12N5/00

PCT International Application Number

N/A

PCT International Filing date

2007-04-23

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA