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Native extracellular matrix preserves mesenchymal stem cell "stemness" and differentiation potential under serum-free culture conditions.

Rakian R, Block TJ, Johnson SM, Marinkovic M, Wu J, Dai Q, Dean DD, Chen XD - Stem Cell Res Ther (2015)

Bottom Line: BM-MSCs, cultured in SFM and treated with BMP-2, retained their differentiation capacity better on BM-ECM than on either of the other two substrates.Our findings indicate that BM-ECM provides a unique microenvironment that supports the colony-forming ability of MSCs in SFM and preserves their stem cell properties.The establishment of a robust culture system, combining native tissue-specific ECM and SFM, provides an avenue for preparing significant numbers of potent MSCs for cell-based therapies in patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229-3900, USA. rakianr@livemail.uthscsa.edu.

ABSTRACT

Introduction: Bone marrow-derived mesenchymal stem cells (BM-MSCs) for clinical use should not be grown in media containing fetal bovine serum (FBS), because of serum-related concerns over biosafety and batch-to-batch variability. Previously, we described the preparation and use of a cell-free native extracellular matrix (ECM) made by bone marrow cells (BM-ECM) which preserves stem cell properties and enhances proliferation. Here, we compare colony-forming ability and differentiation of MSCs cultured on BM-ECM with a commercially available matrix (CELLstart™) and tissue culture plastic (TCP) under serum-free conditions.

Methods: Primary MSCs from freshly isolated bone marrow-derived mononuclear cells or passaged MSCs (P1) were grown in serum-containing (SCM) or serum-free (SFM) media on BM-ECM, CELLstart™, or TCP substrates. Proliferation, cell composition (phenotype), colony-forming unit replication, and bone morphogenetic protein-2 (BMP-2) responsiveness were compared among cells maintained on the three substrates.

Results: Proliferation of primary BM-MSCs was significantly higher in SCM than SFM, irrespectively of culture substrate, suggesting that the expansion of these cells requires SCM. In contrast, passaged cells cultured on BM-ECM or CELLstart™ in SFM proliferated to nearly the same extent as cells in SCM. However, morphologically, those on BM-ECM were smaller and more aligned, slender, and long. Cells grown for 7 days on BM-ECM in SFM were 20-40 % more positive for MSC surface markers than cells cultured on CELLstart™. Cells cultured on TCP contained the smallest number of cells positive for MSC markers. MSC colony-forming ability in SFM, as measured by CFU-fibroblasts, was increased 10-, 9-, and 2-fold when P1 cells were cultured on BM-ECM, CELLstart™, and TCP, respectively. Significantly, CFU-adipocyte and -osteoblast replication of cells grown on BM-ECM was dramatically increased over those on CELLstart™ (2X) and TCP (4-7X). BM-MSCs, cultured in SFM and treated with BMP-2, retained their differentiation capacity better on BM-ECM than on either of the other two substrates.

Conclusions: Our findings indicate that BM-ECM provides a unique microenvironment that supports the colony-forming ability of MSCs in SFM and preserves their stem cell properties. The establishment of a robust culture system, combining native tissue-specific ECM and SFM, provides an avenue for preparing significant numbers of potent MSCs for cell-based therapies in patients.

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Phenotypic expression of MSC surface markers after culture in SFM for 4 and 7 days. Early passage (P1) cultures of BM-MSCs were grown on TCP, BM-ECM, or CELLstart™ in SFM. Phenotypic expression of MSC-associated markers (SSEA-4, CD73, CD90, CD105, and CD146) was assessed by using flow cytometry. a Single-cell suspensions, derived from 7-day cultures on CELLstart™ or BM-ECM, were analyzed by fluorescence-activated cell sorting. In the top panel, dot plots of the cell distribution are shown. Relatively smaller cells are found in “range a” (CELLstart: approximately 30 %; BM-ECM: 62 %), whereas relatively larger cells are found in “range b” (CELLstart: approximately 35 %; BM-ECM: 7 %). In the lower panel, histograms represent the expression of the indicated markers. Cells were stained with primary non-specific antibody (isotype, IgG) as negative controls (gray peaks). b P1 cultures of BM-MSCs were grown on the three culture surfaces for 4 (left panel) and 7 (right panel) days in SFM. The number of positive cells expressing each marker was determined as a percentage of the total cell population (also see Table 1). Mean ± standard deviation was calculated from three independent experiments. *P < 0.05 versus CELLstart™. BM-ECM bone marrow-derived extracellular matrix, BM-MSC bone marrow-derived mesenchymal stem cell, CD, cluster of differentiation/determinants, FSC forward scatter, MSC mesenchymal stem cell, P1 passage 1, SFM serum-free media, SSC side scatter, SSEA-4 stage-specific embryonic antigen-4, TCP tissue culture plastic
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Fig4: Phenotypic expression of MSC surface markers after culture in SFM for 4 and 7 days. Early passage (P1) cultures of BM-MSCs were grown on TCP, BM-ECM, or CELLstart™ in SFM. Phenotypic expression of MSC-associated markers (SSEA-4, CD73, CD90, CD105, and CD146) was assessed by using flow cytometry. a Single-cell suspensions, derived from 7-day cultures on CELLstart™ or BM-ECM, were analyzed by fluorescence-activated cell sorting. In the top panel, dot plots of the cell distribution are shown. Relatively smaller cells are found in “range a” (CELLstart: approximately 30 %; BM-ECM: 62 %), whereas relatively larger cells are found in “range b” (CELLstart: approximately 35 %; BM-ECM: 7 %). In the lower panel, histograms represent the expression of the indicated markers. Cells were stained with primary non-specific antibody (isotype, IgG) as negative controls (gray peaks). b P1 cultures of BM-MSCs were grown on the three culture surfaces for 4 (left panel) and 7 (right panel) days in SFM. The number of positive cells expressing each marker was determined as a percentage of the total cell population (also see Table 1). Mean ± standard deviation was calculated from three independent experiments. *P < 0.05 versus CELLstart™. BM-ECM bone marrow-derived extracellular matrix, BM-MSC bone marrow-derived mesenchymal stem cell, CD, cluster of differentiation/determinants, FSC forward scatter, MSC mesenchymal stem cell, P1 passage 1, SFM serum-free media, SSC side scatter, SSEA-4 stage-specific embryonic antigen-4, TCP tissue culture plastic

Mentions: P1 cells that had been cultured on TCP, BM-ECM, and CELLstart™ in SFM for 4 and 7 days were analyzed for cell number and a panel of MSC surface markers (SSEA-4, CD73, CD90, CD105, and CD146). Although cells cultured for 4 days on BM-ECM showed a trend toward greater numbers of MSCs, it never attained statistical significance. In contrast, after 7 days in culture on BM-ECM, the absolute number of cells and the percent positive for MSC markers were significantly increased over cultures on CELLstart™ (Table 1 and Fig. 4b). Dot plots for cell scatter (Fig. 4a) revealed a different cell distribution after culture on CELLstart™ versus BM-ECM (small cells: approximately 30 % versus approximately 62 %; large cells: approximately 35 % versus approximately 7 %, respectively). After both 4 and 7 days, cultures on TCP contained the smallest number of cells and the lowest percent positive for MSC markers by FACS analysis.Table 1


Native extracellular matrix preserves mesenchymal stem cell "stemness" and differentiation potential under serum-free culture conditions.

Rakian R, Block TJ, Johnson SM, Marinkovic M, Wu J, Dai Q, Dean DD, Chen XD - Stem Cell Res Ther (2015)

Phenotypic expression of MSC surface markers after culture in SFM for 4 and 7 days. Early passage (P1) cultures of BM-MSCs were grown on TCP, BM-ECM, or CELLstart™ in SFM. Phenotypic expression of MSC-associated markers (SSEA-4, CD73, CD90, CD105, and CD146) was assessed by using flow cytometry. a Single-cell suspensions, derived from 7-day cultures on CELLstart™ or BM-ECM, were analyzed by fluorescence-activated cell sorting. In the top panel, dot plots of the cell distribution are shown. Relatively smaller cells are found in “range a” (CELLstart: approximately 30 %; BM-ECM: 62 %), whereas relatively larger cells are found in “range b” (CELLstart: approximately 35 %; BM-ECM: 7 %). In the lower panel, histograms represent the expression of the indicated markers. Cells were stained with primary non-specific antibody (isotype, IgG) as negative controls (gray peaks). b P1 cultures of BM-MSCs were grown on the three culture surfaces for 4 (left panel) and 7 (right panel) days in SFM. The number of positive cells expressing each marker was determined as a percentage of the total cell population (also see Table 1). Mean ± standard deviation was calculated from three independent experiments. *P < 0.05 versus CELLstart™. BM-ECM bone marrow-derived extracellular matrix, BM-MSC bone marrow-derived mesenchymal stem cell, CD, cluster of differentiation/determinants, FSC forward scatter, MSC mesenchymal stem cell, P1 passage 1, SFM serum-free media, SSC side scatter, SSEA-4 stage-specific embryonic antigen-4, TCP tissue culture plastic
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Fig4: Phenotypic expression of MSC surface markers after culture in SFM for 4 and 7 days. Early passage (P1) cultures of BM-MSCs were grown on TCP, BM-ECM, or CELLstart™ in SFM. Phenotypic expression of MSC-associated markers (SSEA-4, CD73, CD90, CD105, and CD146) was assessed by using flow cytometry. a Single-cell suspensions, derived from 7-day cultures on CELLstart™ or BM-ECM, were analyzed by fluorescence-activated cell sorting. In the top panel, dot plots of the cell distribution are shown. Relatively smaller cells are found in “range a” (CELLstart: approximately 30 %; BM-ECM: 62 %), whereas relatively larger cells are found in “range b” (CELLstart: approximately 35 %; BM-ECM: 7 %). In the lower panel, histograms represent the expression of the indicated markers. Cells were stained with primary non-specific antibody (isotype, IgG) as negative controls (gray peaks). b P1 cultures of BM-MSCs were grown on the three culture surfaces for 4 (left panel) and 7 (right panel) days in SFM. The number of positive cells expressing each marker was determined as a percentage of the total cell population (also see Table 1). Mean ± standard deviation was calculated from three independent experiments. *P < 0.05 versus CELLstart™. BM-ECM bone marrow-derived extracellular matrix, BM-MSC bone marrow-derived mesenchymal stem cell, CD, cluster of differentiation/determinants, FSC forward scatter, MSC mesenchymal stem cell, P1 passage 1, SFM serum-free media, SSC side scatter, SSEA-4 stage-specific embryonic antigen-4, TCP tissue culture plastic
Mentions: P1 cells that had been cultured on TCP, BM-ECM, and CELLstart™ in SFM for 4 and 7 days were analyzed for cell number and a panel of MSC surface markers (SSEA-4, CD73, CD90, CD105, and CD146). Although cells cultured for 4 days on BM-ECM showed a trend toward greater numbers of MSCs, it never attained statistical significance. In contrast, after 7 days in culture on BM-ECM, the absolute number of cells and the percent positive for MSC markers were significantly increased over cultures on CELLstart™ (Table 1 and Fig. 4b). Dot plots for cell scatter (Fig. 4a) revealed a different cell distribution after culture on CELLstart™ versus BM-ECM (small cells: approximately 30 % versus approximately 62 %; large cells: approximately 35 % versus approximately 7 %, respectively). After both 4 and 7 days, cultures on TCP contained the smallest number of cells and the lowest percent positive for MSC markers by FACS analysis.Table 1

Bottom Line: BM-MSCs, cultured in SFM and treated with BMP-2, retained their differentiation capacity better on BM-ECM than on either of the other two substrates.Our findings indicate that BM-ECM provides a unique microenvironment that supports the colony-forming ability of MSCs in SFM and preserves their stem cell properties.The establishment of a robust culture system, combining native tissue-specific ECM and SFM, provides an avenue for preparing significant numbers of potent MSCs for cell-based therapies in patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229-3900, USA. rakianr@livemail.uthscsa.edu.

ABSTRACT

Introduction: Bone marrow-derived mesenchymal stem cells (BM-MSCs) for clinical use should not be grown in media containing fetal bovine serum (FBS), because of serum-related concerns over biosafety and batch-to-batch variability. Previously, we described the preparation and use of a cell-free native extracellular matrix (ECM) made by bone marrow cells (BM-ECM) which preserves stem cell properties and enhances proliferation. Here, we compare colony-forming ability and differentiation of MSCs cultured on BM-ECM with a commercially available matrix (CELLstart™) and tissue culture plastic (TCP) under serum-free conditions.

Methods: Primary MSCs from freshly isolated bone marrow-derived mononuclear cells or passaged MSCs (P1) were grown in serum-containing (SCM) or serum-free (SFM) media on BM-ECM, CELLstart™, or TCP substrates. Proliferation, cell composition (phenotype), colony-forming unit replication, and bone morphogenetic protein-2 (BMP-2) responsiveness were compared among cells maintained on the three substrates.

Results: Proliferation of primary BM-MSCs was significantly higher in SCM than SFM, irrespectively of culture substrate, suggesting that the expansion of these cells requires SCM. In contrast, passaged cells cultured on BM-ECM or CELLstart™ in SFM proliferated to nearly the same extent as cells in SCM. However, morphologically, those on BM-ECM were smaller and more aligned, slender, and long. Cells grown for 7 days on BM-ECM in SFM were 20-40 % more positive for MSC surface markers than cells cultured on CELLstart™. Cells cultured on TCP contained the smallest number of cells positive for MSC markers. MSC colony-forming ability in SFM, as measured by CFU-fibroblasts, was increased 10-, 9-, and 2-fold when P1 cells were cultured on BM-ECM, CELLstart™, and TCP, respectively. Significantly, CFU-adipocyte and -osteoblast replication of cells grown on BM-ECM was dramatically increased over those on CELLstart™ (2X) and TCP (4-7X). BM-MSCs, cultured in SFM and treated with BMP-2, retained their differentiation capacity better on BM-ECM than on either of the other two substrates.

Conclusions: Our findings indicate that BM-ECM provides a unique microenvironment that supports the colony-forming ability of MSCs in SFM and preserves their stem cell properties. The establishment of a robust culture system, combining native tissue-specific ECM and SFM, provides an avenue for preparing significant numbers of potent MSCs for cell-based therapies in patients.

Show MeSH
Related in: MedlinePlus