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Cell and Tissue Research

, Volume 350, Issue 1, pp 55–68 | Cite as

Generation of mesenchymal stem cell lines from murine bone marrow

  • P. Sreejit
  • K. B. Dilip
  • R. S. VermaEmail author
Regular Article

Abstract

Mesenchymal stem cells (MSC), because of their multipotency and ease of purification and amplification, are an ideal stem cell source for cell therapies. Bone-marrow-derived stem cells (BMSC) can be used to develop MSC-like immortalized cell lines with large proliferation and differentiation potentialities. Their immortalized status prevents the maintenance of MSC function and characters; this can be negated by modifying the isolation and maintenance protocol. Adult murine BMSC were isolated and maintained in media without additional growth factors together with passage-dependent reseeding following trypsinization. Cells maintained over 25 passages were considered as putative cell lines and characterized. The phenotypic and genotypic characteristics and multilineage differentiation potential of the cells were assessed by morphological, phenotypic, and molecular assays at various passages. The putative BMSC cell lines showed the characteristics of MSC and were able to maintain these characteristics, even after immortalization. The phenotypic data demonstrated difference among two cell lines; this was further validated by the difference in their multilineage differentiation potential following specific induction. More importantly, no changes were observed in the genotypic level in comparison with control cells, even after more than 50 passages. Our protocol thus advances the isolation and maintenance of BMSC and the development of putative BMSC cell lines that maintain characteristics of MSC, including multilineage differentiation potential, after more than 40 passages.

Keywords

Stem cells Cell lines Mesenchymal stem cell characterization Differentiation induction Mouse (Swiss albino) 

Supplementary material

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Supplementary Fig. 1

Phase-contrast microscopy of cardiomyogenic differentiation. 5-Azacytidine-induced BMSC grown on 0.2% gelatin-coated plates, 30 days after initial cardiomyogenic induction. a, d Images of uninduced and induced control BMSC, respectively, at passage 10 (Pn 10) grown on gelatin-coated plates (30th day). b, e Images of differentiated putative BMSC cell line SRMSC-3 (Pn 30 and 60, respectively) grown on gelatin-coated plates following chemical induction (30th day). c, f Images of differentiated putative BMSC cell line SRMSC-4 (Pn 30 and 60, respectively) grown on gelatin-coated plates following chemical induction (30th day). Bars 100 μm. (JPEG 36 kb)

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Supplementary Fig. 2

Immunocytochemistry (ICC) of differentiated BMSC cell lines. Images of ICC analysis on induced BMSC grown on 0.2% gelatin-coated plates, 30 days after initial cardiomyogenic induction. a, b Merged images of CD 29 and GATA-4 expression with Hoechst 33342 nuclear counterstaining in uninduced and induced control BMSC (Pn 10), respectively, grown on gelatin-coated plates (30th day). c, d Merged images of CD 29 and GATA-4 expression with Hoechst 33342 nuclear counterstaining in induced cell lines SRMSC-3 and SRMSC-4 (Pn 30), respectively, following initial chemical induction (30th day). e, f Merged image of CD 106 expression with Hoechst 33342 nuclear counterstaining in uninduced and induced control BMSC (Pn 10), respectively, grown on gelatin-coated plates (30th day). g, h Merged images of CD 106 expression with Hoechst 33342 nuclear counterstaining in induced SRMSC-3 and SRMSC-4 (Pn 30), respectively, following initial chemical induction (30th day). Bars 100 μm (JPEG 28 kb)

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Supplementary Fig. 3

RT-PCR analysis of differentiated BMSC cell lines. Gene expression analysis of 5-azacytidine-induced BMSC cell lines grown on 0.2% gelatin-coated plates after 30 days of initial treatment, with uninduced cells for comparison (Uninduced 3 SRMSC-3 without 5-azacytidine induction, Uninduced 4 SRMSC-4 without 5-azacytidine induction, Induced 3 SRMSC-3 with 5 μM 5-azacytidine induction, Induced 4 SRMSC-4 with 5 μM 5-azacytidine induction). (JPEG 19 kb)

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High resolution image (TIFF 222 kb)
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Supplementary Fig. 4

Cardiomyogenic differentiation efficacy of induced putative BMSC cell lines. Representative fluorescent image of DiI-Hoechst 33342 staining of 5 μM 5-azacytidine-induced BMSC cell lines grown on 0.2% gelatin-coated plates after 30 days of the initial chemical induction. a Uninduced control BMSC at passage 10 (Pn 10) without 5-azacytidine treatment. b Induced control BMSC (Pn 10). c, d Induced SRMSC-3 (Pn 30 and 60, respectively). e, f Induced SRMSC-4 (Pn 30 and 60, respectively). Bars 100 μm. (JPEG 45 kb)

441_2012_1458_MOESM4_ESM.tif (2.2 mb)
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441_2012_1458_MOESM5_ESM.doc (34 kb)
ESM 1 (DOC 33 kb)
441_2012_1458_MOESM6_ESM.doc (36 kb)
ESM 2 (DOC 35 kb)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  1. 1.Stem Cell & Molecular Biology Laboratory (201), Bhupat and Jyoti Mehta School of Biosciences, Department of BiotechnologyIndian Institute of Technology MadrasChennaiIndia

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