DIFFERENTIATION OF ADIPOSE STROMAL CELLS INTO OSTEOBLASTS AND USES THEREOF
The invention provides methods and compositions for differentiating stromal cells from adipose tissue into cells having osteoblastic properties, and methods for improving a subject's bone structure. The methods comprise culturing stromal cells from adipose tissue in β-glycerophosphate and ascorbic acid and/or ascorbate-2-phosphate for a time sufficient to allow differentiation of said cells into osteoblasts. Such methods and compositions are useful in the production of osteoblasts for autologous transplantation into bone at a surgical site or injury. The compositions comprise adipose stromal cells, a medium capable of supporting the growth of fibroblasts and amounts of β-glycerophosphate and ascorbic acid and/or ascorbic-2-phosphate sufficient to induce the differentiation of said stromal cells into osteoblasts. The invention further provides methods of identifying compounds that affect osteoblast differentiation. Such compounds are useful in the study of bone development and in the treatment of bone disorders, including bone fractures and osteoporosis.
1 . A method of differentiating adipose stromal cells into osteoblasts, comprising: culturing said cells in a composition comprising a medium capable of supporting the growth of fibroblasts and differentiation inducing amounts of β-glycerophosphate and ascorbic acid and/or ascorbic-2-phosphate.
2 . The method of claim 1 , wherein said amounts are about 2-20 mM β-glycerophosphate and about 20-75 μM ascorbic acid and/or ascorbic-2-phosphate.
3 . The method of claim 2 wherein said amounts are about 5-15 mM β-glycerophosphate and about 40-60 μM ascorbic acid and/or ascorbic-2-phosphate.
4 . The method of claim 3 , wherein said amounts are about 10 mM β-glycerophosphate and about 50 μM ascorbic acid and/or ascorbic-2-phosphate.
5 . The method of claim 1 , wherein said medium is selected from the group consisting of: DMEM, aMEM and BME.
6 . The method of claim 1 , wherein said medium further comprises about 5-20% fetal calf serum.
7 . The method of claim 1 , wherein said medium further comprises one or more bone morphogenetic proteins.
8 . The method of claim 1 wherein said cells are mammalian.
9 . The method of claim 8 wherein said cells are human.
10 . A method of improving a subject's bone structure, comprising:
a. culturing adipose stromal cells in a composition which comprises a medium capable of supporting the growth of fibroblasts and differentiation inducing amounts of β-glycerophosphate and ascorbic acid and/or ascorbic-2-phosphate; and
b. introducing said osteoblasts into a surgery or fracture site of said subject.
11 . The method of claim 10 , wherein said amounts are about 2-20 mM β-glycerophosphate and about 20-75 μM ascorbic acid and/or ascorbic-2-phosphate.
12 . The method of claim 11 , wherein said amounts are about 5-15 mM β-glycerophosphate and about 40-60 μM ascorbic acid and/or ascorbic-2-phosphate.
13 . The method of claim 12 , wherein said amounts are about 10 mM β-glycerophosphate and about 50 μM ascorbic acid and/or ascorbic-2-phosphate.
14 . The method of claim 10 , wherein said adipose stromal cells are isolated from said subject.
15 . The method of claim 10 , wherein said medium is selected from the group consisting of: DMEM, aMEM and BME.
16 . The method of claim 10 , wherein said medium further comprises about 5-20% fetal calf serum.
17 . The method of claim 10 , wherein said medium further comprises one or more bone morphogenetic proteins.
18 . The method of claim 10 , wherein said subject is mammalian.
19 . The method of claim 18 , wherein said subject is human.
20 . The method of claim 10 , wherein said osteoblasts are introduced in admixture with a composition useful in the repair of bone wounds, bone defects and/or bone disorders.
21 . The method of claim 10 , wherein a nucleotide sequence of interest is introduced into said adipose stromal cells or said osteoblasts.