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Isolation, selection and culture methods to enhance clonogenicity of mouse bone marrow derived mesenchymal stromal cell precursors.

Baustian C, Hanley S, Ceredig R - Stem Cell Res Ther (2015)

Bottom Line: By fluorescence activated cell sorting of CD45(-)/Ter119(-) mBM stroma based on Sca-1 expression and expansion in hypoxic conditions, we show that Sca-1(+) cells had higher CFU-F frequencies and showed enhanced proliferation compared with Sca-1(-) cells.Functional investigations demonstrated that these MSC clones inhibited T-lymphocyte proliferation.By positive selection using a combination of antibodies to Sca-1, CD90 and PDGFRα and culturing in hypoxia, we have found a subpopulation of BM cells from C57Bl/6 mice with a CFU-F cloning efficiency of 1/4.

View Article: PubMed Central - PubMed

Affiliation: Regenerative Medicine Institute, National Centre for Biomedical Engineering Science and School of Medicine, National University of Ireland, Galway, Ireland. c.baustian1@nuigalway.ie.

ABSTRACT

Introduction: Conventionally cultured mouse bone marrow mesenchymal stromal cells (mBM-MSC) are a heterogeneous population that often initially contain contaminating haematopoietic cells. Variability in isolation methods, culture protocols and the lack of specific mBM MSC markers might explain this heterogeneity. The aim of this study is to optimise the isolation, culture conditions and selection of mBM-MSC.

Methods: Mouse BM-MSCs were isolated from crushed long bones (cBM) or flushed bone marrow (fBM) from 6-8 week old C57Bl/6 mice. These subpopulations were analysed by flow cytometry using commonly used mBM-MSC cell surface marker, e.g. Sca-1, CD29 and CD44. Cells were cultured and expanded in vitro in hypoxic conditions of either 2 % or 5 % oxygen. Cell sorting and qRT-PCR was used to determine transcript levels of stem cell and lineage related genes in individual subpopulations.

Results: During early passaging not only do contaminating haematopoietic cells disappear, but there is a change in the phenotype of mBM-MSC affecting particularly CD44 and Sca-1 expression. By fluorescence activated cell sorting of CD45(-)/Ter119(-) mBM stroma based on Sca-1 expression and expansion in hypoxic conditions, we show that Sca-1(+) cells had higher CFU-F frequencies and showed enhanced proliferation compared with Sca-1(-) cells. As evaluated by in vitro assays and qRT-PCR, these cells presented in vitro tri-lineage differentiation along osteocyte, chondrocyte, and adipocyte lineages. Finally, by prospective isolation of Sca-1(+)PDGFRα(+)CD90(+) cells we have isolated mBM-MSC on a single cell level, achieving a CFU-F frequency of 1/4. Functional investigations demonstrated that these MSC clones inhibited T-lymphocyte proliferation.

Conclusion: By positive selection using a combination of antibodies to Sca-1, CD90 and PDGFRα and culturing in hypoxia, we have found a subpopulation of BM cells from C57Bl/6 mice with a CFU-F cloning efficiency of 1/4. To our knowledge these results represent the highest frequencies of mouse MSC cloning from C57Bl/6 mice yet reported.

No MeSH data available.


Related in: MedlinePlus

Use of Sca-1 as a selection marker for MSCs. a Sca-1+ and sca-1− from cBM were cultured in osteogenic (upper), adipogenic (middle) and chondrogenic (lower) differentiation media followed by Alizarin Red S (upper), Oil Red O (middle) or Safranin O (lower) staining. Osteogenic and adipogenic images, bar = 100 μm; chondrogenic images, bar = 200 μm. b Chondrogenic pellet cultures from the indicated subpopulations assayed for S-GAG content. c Fold change of Sca-1+ relative to Sca-1− expression of stem and lineage-specific gene transcripts in freshly isolated and cultured (passage 2 (p2)) cells. Fold change of relative expression of transcripts associated with chondrogenesis in d freshly isolated Sca-1− versus Sca-1+ sorted cells and e cultured articular chondrocytes versus cultured Sca-1− cells. Data are the mean ± SD of at least three independent experiments. **p <0.01, Student’s t test. s-GAG sulfated glycosaminoglycans
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Fig4: Use of Sca-1 as a selection marker for MSCs. a Sca-1+ and sca-1− from cBM were cultured in osteogenic (upper), adipogenic (middle) and chondrogenic (lower) differentiation media followed by Alizarin Red S (upper), Oil Red O (middle) or Safranin O (lower) staining. Osteogenic and adipogenic images, bar = 100 μm; chondrogenic images, bar = 200 μm. b Chondrogenic pellet cultures from the indicated subpopulations assayed for S-GAG content. c Fold change of Sca-1+ relative to Sca-1− expression of stem and lineage-specific gene transcripts in freshly isolated and cultured (passage 2 (p2)) cells. Fold change of relative expression of transcripts associated with chondrogenesis in d freshly isolated Sca-1− versus Sca-1+ sorted cells and e cultured articular chondrocytes versus cultured Sca-1− cells. Data are the mean ± SD of at least three independent experiments. **p <0.01, Student’s t test. s-GAG sulfated glycosaminoglycans

Mentions: Next we examined the differentiation capacity of Sca-1+ and Sca-1− cells to the osteocyte, adipocyte and chondrocyte lineages. After two passages, cells were transferred to the corresponding differentiation conditions, and after a further 14 days cells were analysed for osteocyte and adipocyte differentiation; chondrocyte differentiation was measured after 21 days. As shown in Fig. 4a, both Sca-1+ and Sca-1− cells differentiated along the osteocyte and adipocyte lineages; however, Sca-1− cells showed increased differentiation to chondrocytes as demonstrated by Safranin-O staining for proteoglycans (Fig. 4a, lower panel). This was confirmed by quantification for sulfated glycosaminoglycans (S-GAG) with an approximately threefold greater S-GAG content in Sca-1− cells (Fig. 4b). No significant difference between the two subpopulations in osteogenic or adipogenic differentiation was found (data not shown).Fig. 4


Isolation, selection and culture methods to enhance clonogenicity of mouse bone marrow derived mesenchymal stromal cell precursors.

Baustian C, Hanley S, Ceredig R - Stem Cell Res Ther (2015)

Use of Sca-1 as a selection marker for MSCs. a Sca-1+ and sca-1− from cBM were cultured in osteogenic (upper), adipogenic (middle) and chondrogenic (lower) differentiation media followed by Alizarin Red S (upper), Oil Red O (middle) or Safranin O (lower) staining. Osteogenic and adipogenic images, bar = 100 μm; chondrogenic images, bar = 200 μm. b Chondrogenic pellet cultures from the indicated subpopulations assayed for S-GAG content. c Fold change of Sca-1+ relative to Sca-1− expression of stem and lineage-specific gene transcripts in freshly isolated and cultured (passage 2 (p2)) cells. Fold change of relative expression of transcripts associated with chondrogenesis in d freshly isolated Sca-1− versus Sca-1+ sorted cells and e cultured articular chondrocytes versus cultured Sca-1− cells. Data are the mean ± SD of at least three independent experiments. **p <0.01, Student’s t test. s-GAG sulfated glycosaminoglycans
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4549076&req=5

Fig4: Use of Sca-1 as a selection marker for MSCs. a Sca-1+ and sca-1− from cBM were cultured in osteogenic (upper), adipogenic (middle) and chondrogenic (lower) differentiation media followed by Alizarin Red S (upper), Oil Red O (middle) or Safranin O (lower) staining. Osteogenic and adipogenic images, bar = 100 μm; chondrogenic images, bar = 200 μm. b Chondrogenic pellet cultures from the indicated subpopulations assayed for S-GAG content. c Fold change of Sca-1+ relative to Sca-1− expression of stem and lineage-specific gene transcripts in freshly isolated and cultured (passage 2 (p2)) cells. Fold change of relative expression of transcripts associated with chondrogenesis in d freshly isolated Sca-1− versus Sca-1+ sorted cells and e cultured articular chondrocytes versus cultured Sca-1− cells. Data are the mean ± SD of at least three independent experiments. **p <0.01, Student’s t test. s-GAG sulfated glycosaminoglycans
Mentions: Next we examined the differentiation capacity of Sca-1+ and Sca-1− cells to the osteocyte, adipocyte and chondrocyte lineages. After two passages, cells were transferred to the corresponding differentiation conditions, and after a further 14 days cells were analysed for osteocyte and adipocyte differentiation; chondrocyte differentiation was measured after 21 days. As shown in Fig. 4a, both Sca-1+ and Sca-1− cells differentiated along the osteocyte and adipocyte lineages; however, Sca-1− cells showed increased differentiation to chondrocytes as demonstrated by Safranin-O staining for proteoglycans (Fig. 4a, lower panel). This was confirmed by quantification for sulfated glycosaminoglycans (S-GAG) with an approximately threefold greater S-GAG content in Sca-1− cells (Fig. 4b). No significant difference between the two subpopulations in osteogenic or adipogenic differentiation was found (data not shown).Fig. 4

Bottom Line: By fluorescence activated cell sorting of CD45(-)/Ter119(-) mBM stroma based on Sca-1 expression and expansion in hypoxic conditions, we show that Sca-1(+) cells had higher CFU-F frequencies and showed enhanced proliferation compared with Sca-1(-) cells.Functional investigations demonstrated that these MSC clones inhibited T-lymphocyte proliferation.By positive selection using a combination of antibodies to Sca-1, CD90 and PDGFRα and culturing in hypoxia, we have found a subpopulation of BM cells from C57Bl/6 mice with a CFU-F cloning efficiency of 1/4.

View Article: PubMed Central - PubMed

Affiliation: Regenerative Medicine Institute, National Centre for Biomedical Engineering Science and School of Medicine, National University of Ireland, Galway, Ireland. c.baustian1@nuigalway.ie.

ABSTRACT

Introduction: Conventionally cultured mouse bone marrow mesenchymal stromal cells (mBM-MSC) are a heterogeneous population that often initially contain contaminating haematopoietic cells. Variability in isolation methods, culture protocols and the lack of specific mBM MSC markers might explain this heterogeneity. The aim of this study is to optimise the isolation, culture conditions and selection of mBM-MSC.

Methods: Mouse BM-MSCs were isolated from crushed long bones (cBM) or flushed bone marrow (fBM) from 6-8 week old C57Bl/6 mice. These subpopulations were analysed by flow cytometry using commonly used mBM-MSC cell surface marker, e.g. Sca-1, CD29 and CD44. Cells were cultured and expanded in vitro in hypoxic conditions of either 2 % or 5 % oxygen. Cell sorting and qRT-PCR was used to determine transcript levels of stem cell and lineage related genes in individual subpopulations.

Results: During early passaging not only do contaminating haematopoietic cells disappear, but there is a change in the phenotype of mBM-MSC affecting particularly CD44 and Sca-1 expression. By fluorescence activated cell sorting of CD45(-)/Ter119(-) mBM stroma based on Sca-1 expression and expansion in hypoxic conditions, we show that Sca-1(+) cells had higher CFU-F frequencies and showed enhanced proliferation compared with Sca-1(-) cells. As evaluated by in vitro assays and qRT-PCR, these cells presented in vitro tri-lineage differentiation along osteocyte, chondrocyte, and adipocyte lineages. Finally, by prospective isolation of Sca-1(+)PDGFRα(+)CD90(+) cells we have isolated mBM-MSC on a single cell level, achieving a CFU-F frequency of 1/4. Functional investigations demonstrated that these MSC clones inhibited T-lymphocyte proliferation.

Conclusion: By positive selection using a combination of antibodies to Sca-1, CD90 and PDGFRα and culturing in hypoxia, we have found a subpopulation of BM cells from C57Bl/6 mice with a CFU-F cloning efficiency of 1/4. To our knowledge these results represent the highest frequencies of mouse MSC cloning from C57Bl/6 mice yet reported.

No MeSH data available.


Related in: MedlinePlus