Heparan sulfate enhances the self-renewal and therapeutic potential of mesenchymal stem cells from human adult bone marrow - PubMed (original) (raw)

. 2012 Jul 20;21(11):1897-910.

doi: 10.1089/scd.2011.0367. Epub 2012 Jan 18.

Christian Dombrowski, Bina Rai, Zophia X H Lim, Ian Lee Hock Hin, David A Rider, Gary S Stein, Wanjin Hong, Andre J van Wijnen, James H Hui, Victor Nurcombe, Simon M Cool

Affiliations

Heparan sulfate enhances the self-renewal and therapeutic potential of mesenchymal stem cells from human adult bone marrow

Torben Helledie et al. Stem Cells Dev. 2012.

Abstract

Insufficient cell number hampers therapies utilizing adult human mesenchymal stem cells (hMSCs) and current ex vivo expansion strategies lead to a loss of multipotentiality. Here we show that supplementation with an embryonic form of heparan sulfate (HS-2) can both increase the initial recovery of hMSCs from bone marrow aspirates and increase their ex vivo expansion by up to 13-fold. HS-2 acts to amplify a subpopulation of hMSCs harboring longer telomeres and increased expression of the MSC surface marker stromal precursor antigen-1. Gene expression profiling revealed that hMSCs cultured in HS-2 possess a distinct signature that reflects their enhanced multipotentiality and improved bone-forming ability when transplanted into critical-sized bone defects. Thus, HS-2 offers a novel means for decreasing the expansion time necessary for obtaining therapeutic numbers of multipotent hMSCs without the addition of exogenous growth factors that compromise stem cell fate.

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Figures

FIG. 1.

FIG. 1.

Short-term exposure to heparan sulfate-2 increases the proliferation of human mesenchymal stem cells. (A) Growth of cells exposed to 160 ng/mL heparan sulfate (HS-2) over 8 days; (B) their morphology under an inverted phase contrast microscope, bar=200 μm. (C) Apoptotic levels after 8 days exposure to HS-2. (D) Incorporation of BrdU during short-term exposure to HS-2. (E) Proportion of cells in S/G2/M phases of the cell cycle when cultured in HS-2. (F) HS-2 sustains the growth of human mesenchymal stem cells (hMSCs) in decreasing concentrations of FCS as determined by WST-1 assay. Error bars represent the standard deviation, _n_=3.

FIG. 2.

FIG. 2.

Supplementation with heparan sulfate-2 supports the expansion of stromal precursor antigen-1+ human mesenchymal stem cells. (A) FACS analysis confirms that early passage hMSCs express high levels of CD73 and CD105 and moderate levels of CD49a and stromal precursor antigen (STRO-1). (B) Continued subculturing in HS-2 (160 ng/mL) increases STRO-1 expression, (C) and the STRO-1Bright+ population. (D) Colony efficiency assays indicated that HS-2-expanded cells form ∼50% more colonies compared with cells cultured in control media. Error bars represent the standard deviation, _n_=3.

FIG. 3.

FIG. 3.

Heparan sulfate-2 enriches a subpopulation of more naïve human mesenchymal stem cells with longer telomeres during continuous subculturing. (A) hMSCs were expanded in control or HS-2 media for 45 days and the cumulative cell number plotted against time. (B) Relative telomere length of hMSCs expanded in control or HS-2 media for 15 population doublings (PD). (C) Expression of surface markers in hMSCs expanded ∼p7 in control or HS-2 media. (D–F) Differentiative potential of hMSCs continuously subcultured for ∼p7 in control or HS-2 media. Expanded cells were then differentiated for 28 days in adipogenic (D), osteogenic (E), or 21 days in chondrogenic media (F). Adipogenesis was measured by Oil-Red-O staining (Scale bar=1 mm) and quantitative PCR for CCAAT/enhancer binding protein-a (C/EBPa) and adipocyte lipid binding protein (ALBP); osteogenesis was measured by alkaline phosphatase (ALP), alizarin red staining (Scale bar=1 mm), and quantitative PCR for ALP and osteocalcin (OC); and chondrogenesis was measured by H&E and alcian blue staining (Scale bar=500 μm) and quantitative PCR for collagen2a1 (Coll2a) and SOX9. Error bars represent the standard deviation, _n_=3. Color images available online at

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FIG. 4.

FIG. 4.

Heparan sulfate-2 expands single cell hMSC clones that readily undergo multilineage differentiation. (A) Single cell colony formation of hMSCs pre-expanded in HS-2 or control media for 13 population doublings; (B) multilineage differentiation assays of representative clones from these colonies. From top to bottom: phase contrast micrographs of lipid containing cells from adipogenic cultures, alizarin red staining of mineralizing osteogenic cultures, and alcian blue staining of chondrogenic cultures. (Scale bars=200 μm). Error bars represent standard deviation, n= 3. Color images available online at

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FIG. 5.

FIG. 5.

Heparan sulfate-2 protects hMSC cultures against the temporal loss of the stem cell gene signature. (A) Heirarchical clustering and heat map analysis of genes expressed in hMSCs maintained in HS-2 or control media. (B) HS-2 supplementation upregulates genes involved in cell proliferation while downregulating those involved in differentiation. (C) Singular value decomposition of stem cell related genes for hMSC cultures in HS-2 or control media. Cells grown in HS-2 cluster with control cells from earlier cultures. Color images available online at

www.liebertonline.com/scd

FIG. 6.

FIG. 6.

Human MSCs cultured in heparan sulfate-2 retain their immunomodulatory capabilities and augment bone formation when transplanted in vivo. (A) Immunomodulatory activity of hMSCs expanded in control or HS-2 supplemented media. A mixture of stimulatory and reactionary PBMCs from 2 different donors were added to wells at different hMSC:PBMC ratios and the expression of CD3+ Ki67+ assessed by FACS. The positive control represents the maximum number of CD3+ Ki67+ cells obtained in the absence of HS-2- or control-expanded hMSCs. Readings are in duplicate for each experiment and the graph is the mean±standard deviation (SD) of 2 separate experiments. (B) Representative X-ray and micro-computed tomography (μ-CT) images of 8 mm critical-sized nude rat femoral defects treated with transplanted HS-2- or control-expanded hMSCs after 3 and 7 weeks. (C) Percent bone volume in the defect site at weeks 3 and 7. Results are expressed as mean±SD, _n_=6 for each treatment group, per time point. Significance was determined by a two-tailed unpaired _t_-test * p<0.05. (D) Representative cryosections of defects revealing the surviving hMSCs. DAPI stains the nuclei of all cells blue, while the hMSCs prelabeled with QTracker® fluoresce red. (E) Representative histological sections of defects at week 7 stained with H&E and von Kossa. Arrows indicate the edges of host bone. NB: new bone, HB: host bone, BM: bone marrow, SC: scaffold. Scale bar=500 μm. (F) Magnification of representative defects at week 7 showing abundant osteocalcin-positive osteoblasts present within the newly regenerated bone. * indicate the border of osteoblasts slightly away from the scaffold pore. SC, scaffold; BM, bone marrow. Scale bar=100 μm. Color images available online at

www.liebertonline.com/scd

FIG. 7.

FIG. 7.

Exposure to heparan sulfate-2 increases the growth and frequency of colony forming units-fibroblastic in primary bone marrow aspirates and stimulates expansion of the stromal precursor antigen-1 positive subpopulation. (A) Colony growth from 3 different bone marrow donors in control or HS-2-containing media and (B) colony frequency in control and HS-2-containing media. (C) Representative example of surface marker expression from colonies recovered in control or HS-2-containing media. (D) STRO-1 expression from (C); isotype control is indicated by red line, control by green line, and STRO-1 by blue line showing increase in the STRO-1Bright+ population. (E–G) Cumulative growth of hMSCs from 3 different primary bone marrow donors in HS-2 or control supplemented media.

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