Identification and isolation of multipotent cells from non-osteochondral mesenchymal tissue

Abstract

Methods for the identification and isolation of multipotent cells from non-osteochondral mesenchymal tissue. Specifically, this invention relates to an adult multipotent cell or a cell population or composition comprising said cell, isolated from non-osteochondral mesenchymal tissue, characterized in that the cell is positive for the following markers: CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 and because it lacks expression of the following markers: CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 and CD133.

Description

BACKGROUND OF THE INVENTION

[0001] Stem cells show differential characteristics as they are able to sustain themselves and differentiate into one or more cell type. Although research into stem cells and their applications is still in its early stages, adult stem cells in bone marrow have been used in transplants for more than 30 years. Nevertheless, in recent years, stem cell technology has made large advances such that stem cells are currently considered as a promising source of tissue and organs, with an important therapeutic potential for repair and regeneration of tissues.

[0002] The use of stem cells is an alternative therapy for several human diseases, particularly those in which there is a loss of functional cells, including chondral, bone and muscular lesions, neurodegenerative diseases, immunologic rejection, heart disease and skin disorders (see U.S. Pat. Nos. 5,811,094, 5,958,767, 6,328,960, 6,379,953, 6,497,875).

[0003] In addition to cell therapy applications, stem cell...

Examples

example 1

Isolation of Stem Cells from Soft Tissue and Characterization of Surface Markers

[0064] The isolation of multipotent stem cells from soft tissue was performed by selecting those cells with a capacity for proliferation and differentiation, characterized in that they show adhesion to the plastic container of the cell culture. Then, the cells were characterized by monitoring by flow cytometry of the expression of a series of surface markers on the recently isolated cells and during the course of the culture development in vitro.

[0065] The isolation of the multipotent stem cells was carried out from subdermal adipose tissue, obtained by liposuction from three health donors (donors 1, 2 and 3).

[0066] First, the sample from the subdermal adipose tissue was washed with phosphate buffered saline solution (PBS). To achieve destruction of the extracellular matrix and the isolation of the cells, an enzymatic digestion was performed with type II collagenase in saline solution (5 mg / ml) at 37°...

example 2

In Vitro Differentiation of Multipotent Stem Cells from Human Non-Osteochondral Mesenchymal Tissue into Bone Phenotype Cells

[0083] In the differentiation assay, characterized human cells were used. The cells were isolated from the three samples of lipoaspirate analyzed, each corresponding to a healthy donor. A sample of Mesenchymal Stem Cells (MSC) of human bone marrow was used as a the positive control.

[0084] The cells isolated were seeded at a density of 10,000 cells / cm2 onto 6-well plates (one plate per sample), and were incubated in standard culture medium (DMEM, 10% FBS, L-Glutamine 2 mM and antibiotic). After two days of culturing, the culture medium of one of the wells (control) is replaced with fresh medium, and the remaining wells by osteogenesis inducing medium, which contains the standard culture medium with the following added: [0085] Dexamethasone 100 nM, [0086] Ascorbic acid 50 μM; and [0087]β-Glycerophosphate 10 mM.

[0088] The cells are cultured for 3 weeks under no...

example 3

In Vitro Differentiation of Multipotent Stem Cells from Human Non-Osteochondral Mesenchymal Tissue into Muscle Phenotype Cells

[0090] In the differentiation assay, characterized human cells were isolated from the three liposuction samples each corresponding to a healthy donor, as well as a sample of Mesenchymal Stem Cells (MSC) of human bone marrow, which was used as the positive control.

[0091] The cells isolated were seeded at a density of 10,000 cells / cm2 into standard culture medium (DMEM, 10% FBS, L-Glutamine 2 mM and antibiotic). After two days of culturing, the culture medium of one of the wells (control) is replaced with fresh medium, and the remaining wells by myogenesis inducing medium (Wakitani et al., 1995), which contains the standard culture medium with the following added: [0092] Ascorbate-2-phosphate 0.1 mM, [0093] Dexamethasone 0.01 μM, [0094] ITS+1 (Sigma-Aldrich), and [0095] 5-Azacytidine 3 μM.

[0096] After 24 hours, the medium is replaced by standard culture medi...

BACKGROUND OF THE INVENTION

[0001] Stem cells show differential characteristics as they are able to sustain themselves and differentiate into one or more cell type. Although research into stem cells and their applications is still in its early stages, adult stem cells in bone marrow have been used in transplants for more than 30 years. Nevertheless, in recent years, stem cell technology has made large advances such that stem cells are currently considered as a promising source of tissue and organs, with an important therapeutic potential for repair and regeneration of tissues.

[0002] The use of stem cells is an alternative therapy for several human diseases, particularly those in which there is a loss of functional cells, including chondral, bone and muscular lesions, neurodegenerative diseases, immunologic rejection, heart disease and skin disorders (see U.S. Pat. Nos. 5,811,094, 5,958,767, 6,328,960, 6,379,953, 6,497,875).

[0003] In addition to cell therapy applications, stem cell...

Examples

example 1

Isolation of Stem Cells from Soft Tissue and Characterization of Surface Markers

[0064] The isolation of multipotent stem cells from soft tissue was performed by selecting those cells with a capacity for proliferation and differentiation, characterized in that they show adhesion to the plastic container of the cell culture. Then, the cells were characterized by monitoring by flow cytometry of the expression of a series of surface markers on the recently isolated cells and during the course of the culture development in vitro.

[0065] The isolation of the multipotent stem cells was carried out from subdermal adipose tissue, obtained by liposuction from three health donors (donors 1, 2 and 3).

[0066] First, the sample from the subdermal adipose tissue was washed with phosphate buffered saline solution (PBS). To achieve destruction of the extracellular matrix and the isolation of the cells, an enzymatic digestion was performed with type II collagenase in saline solution (5 mg / ml) at 37°...

example 2

In Vitro Differentiation of Multipotent Stem Cells from Human Non-Osteochondral Mesenchymal Tissue into Bone Phenotype Cells

[0083] In the differentiation assay, characterized human cells were used. The cells were isolated from the three samples of lipoaspirate analyzed, each corresponding to a healthy donor. A sample of Mesenchymal Stem Cells (MSC) of human bone marrow was used as a the positive control.

[0084] The cells isolated were seeded at a density of 10,000 cells / cm2 onto 6-well plates (one plate per sample), and were incubated in standard culture medium (DMEM, 10% FBS, L-Glutamine 2 mM and antibiotic). After two days of culturing, the culture medium of one of the wells (control) is replaced with fresh medium, and the remaining wells by osteogenesis inducing medium, which contains the standard culture medium with the following added: [0085] Dexamethasone 100 nM, [0086] Ascorbic acid 50 μM; and [0087]β-Glycerophosphate 10 mM.

[0088] The cells are cultured for 3 weeks under no...

example 3

In Vitro Differentiation of Multipotent Stem Cells from Human Non-Osteochondral Mesenchymal Tissue into Muscle Phenotype Cells

[0090] In the differentiation assay, characterized human cells were isolated from the three liposuction samples each corresponding to a healthy donor, as well as a sample of Mesenchymal Stem Cells (MSC) of human bone marrow, which was used as the positive control.

[0091] The cells isolated were seeded at a density of 10,000 cells / cm2 into standard culture medium (DMEM, 10% FBS, L-Glutamine 2 mM and antibiotic). After two days of culturing, the culture medium of one of the wells (control) is replaced with fresh medium, and the remaining wells by myogenesis inducing medium (Wakitani et al., 1995), which contains the standard culture medium with the following added: [0092] Ascorbate-2-phosphate 0.1 mM, [0093] Dexamethasone 0.01 μM, [0094] ITS+1 (Sigma-Aldrich), and [0095] 5-Azacytidine 3 μM.

[0096] After 24 hours, the medium is replaced by standard culture medi...

Images