Identification and isolation of small CD44-negative mesenchymal stem/progenitor cells from human bone marrow using elutriation and polychromatic flow cytometry - PubMed (original) (raw)

Identification and isolation of small CD44-negative mesenchymal stem/progenitor cells from human bone marrow using elutriation and polychromatic flow cytometry

Sean R R Hall et al. Stem Cells Transl Med. 2013 Aug.

Abstract

The method of isolation of bone marrow (BM) mesenchymal stem/stromal cells (MSCs) is a limiting factor in their study and therapeutic use. MSCs are typically expanded from BM cells selected on the basis of their adherence to plastic, which results in a heterogeneous population of cells. Prospective identification of the antigenic profile of the MSC population(s) in BM that gives rise to cells with MSC activity in vitro would allow the preparation of very pure populations of MSCs for research or clinical use. To address this issue, we used polychromatic flow cytometry and counterflow centrifugal elutriation to identify a phenotypically distinct population of mesenchymal stem/progenitor cells (MSPCs) within human BM. The MSPC activity resided within a population of rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack CD44, an antigen that is highly expressed on culture-expanded MSCs. In culture, these MSPCs adhere to plastic, rapidly proliferate, and acquire CD44 expression. They form colony forming units-fibroblast and are able to differentiate into osteoblasts, chondrocytes, and adipocytes under defined in vitro conditions. Their acquired expression of CD44 can be partially downregulated by treatment with recombinant human granulocyte-colony stimulating factor, a response not found in BM-MSCs derived from conventional plastic adherence methods. These observations indicate that MSPCs within human BM are rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack expression of CD44. These MSPCs give rise to MSCs that have phenotypic and functional properties that are distinct from those of BM-MSCs purified by plastic adherence.

Keywords: Bone marrow; CD44; Elutriation; FACS; Mesenchymal progenitor cells.

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Figures

Figure 1.

Figure 1.

Prospective identification and isolation of small CD45−CD73+CD90+CD105+CD44− human bone marrow (BM) cells that behave as mesenchymal stem/stromal cells in culture using polychromatic flow cytometry/cell sorting. Using a five-antibody/viability marker panel, we identified a subset of small cells within fresh human BM that were negative for CD44−. (A, B): Mononuclear cells were initially displayed on an SSC/FSC color density plot of BM cells (A), which was subgated onto an antigen plot (B) for identification of CD45−7-AAD− (live) cells (gate R1). (C, D): CD45−7-AAD− cells were further subgated to display of a cluster of CD73+CD90+ cells (gate R2) (C), which were then displayed as a quadrant gate (D) to identify the CD105+CD44− subset of cells. The sort window of CD105+CD44− cells is indicated as R3. (E, F): Back-gating of the original CD45−CD73+CD90+CD105+CD44− population revealed their location near the lymphocyte population within the SSC-height/FSC-height color density plot (E), and standard-sized flow cytometric beads confirmed their small size (right) (F). (G): Representative image of typical colony forming unit-fibroblast from CD45−CD73+CD90+CD105+CD44− sorted cells and higher power image of a single colony (×4, right). (H): Fluorescence-activated cell sorting-sorted CD44− cells were expanded in culture and demonstrated trilineage differentiation potential in vitro toward adipocytes detected using Oil Red O stain for lipids (×10, left), osteoblasts detected using alizarin red S stain (middle), and chondroblasts detected using safranin-O stain (right). Similar results were seen in four other BM samples from different donors. Abbreviations: 7-AAD, 7-aminoactinomycin D; FSC, forward scatter; SSC, side scatter.

Figure 2.

Figure 2.

Characteristics of FACS CD44− fraction isolated from lysed bone marrow. (A): Viable nucleated cells obtained from lysed bone marrow from healthy donors. (B): Differential cell count compared with TNCs. (C): Number of sorted CD45−CD73+CD90+CD105+CD44− cells obtained from CD34/CD133-depleted bone marrow. (D): Percentage of CD45−CD73+CD90+CD105+CD44− cells normalized to TNCs. (E): Growth potential of CD45−CD73+CD90+CD105+CD44− cells over 21 days. Data are presented as mean ± SEM, n = 5. Differential cell counts were performed using an automated hematology analyzer (Beckman Coulter). Abbreviations: FACS, fluorescence-activated cell sorting; TNC, total nucleated cells.

Figure 3.

Figure 3.

Characteristics of elutriated fractions from lysed bone marrow (BM). (A–D): Distribution of viable nucleated cells (A) and percentage of lymphocytes (B), monocytes (C), and granulocytes (D) recovered from the various fractions of lysed BM. (E): Quantification of FACS-sorted CD44− cells from the four fractions. Peak recovery was found in fraction 90. (F): The percentage of rare CD45−CD73+CD90+CD105+CD44− cells from the various fractions when normalized to TNCs demonstrated that fraction 90 contained the bulk of cells. CD45−CD73+CD90+CD105+CD44− cells from fraction 90 were sorted, and the cumulative growth curve (G) was calculated over 21 days after initial plating, calculated as day 0. Data are presented as mean ± SEM, n = 5. *, p < .05 versus fractions 100–110 and >110, using one-way analysis of variance followed by the Newman-Keuls multiple comparison test. Differential cell counts were performed using an automated hematology analyzer (Beckman Coulter). Abbreviations: FACS, fluorescence-activated cell sorting; TNC, total nucleated cells.

Figure 4.

Figure 4.

Fractionation of human bone marrow (BM) using counterflow centrifugal elutriation combined with fluorescence-activated cell sorting using the five-antibody/viability marker panel shows that CD45−CD73+CD90+CD105+CD44− MSPCs elute as small cells (70 and 90 ml/minute). (A–D): Representative display of SSC/FSC color density plots of the elutriated fractions 50/70 (A), 90 (B), 100–110 (C), and >110 (D). Display of CD45/7-AAD density plot was used to identify CD45−7-AAD− (live) cells (gate R3), which was subgated onto a CD73/CD90 antigen plot and identified a cluster of live CD45−CD73+CD90+cells (gate R4), which was further subgated onto a CD105/CD44 antigen plot. Shown is the display of CD105+CD44− on live CD45−CD73+CD90+ gated cells. The sort window shows a cluster of CD105+CD44− cells (R5). The sorted live CD45−CD73+CD90+CD105+CD44− cells (gate R5) are distributed close to the location of lymphocytes when back-gated onto an SSC-height/FSC-height dot plot (far right). Representative images from one experiment are shown. Similar results were seen in four other BM samples from different donors. Abbreviations: 7-AAD, 7-aminoactinomycin D; Frac, fraction; FSC, forward scatter; SSC, side scatter.

Figure 5.

Figure 5.

Immunophenotypic analysis of elutriated CD44− MSPCs. Single-color flow cytometric analysis of selected cell surface proteins on passage 3 culture-expanded CD45−CD73+CD90+CD105+CD44− cells (top panels in each row, FACS) compared with passage 3 donor-matched mesenchymal stem/stromal cells isolated using conventional methods (bottom panels in each row, Ficoll). Shown are representative images from one experiment. Similar results were seen in four other bone marrow samples from different donors. Blue line, isotype control; red line, primary antibody. Abbreviation: FACS, fluorescence-activated cell sorting.

Figure 6.

Figure 6.

Detection of CD45−CD73+CD90+CD105+CD44− cells in G-CSF-mobilized peripheral blood, as previously defined in the bone marrow. (A): Representative display of flow cytometric density plots from fraction 90. Mononuclear cells were initially displayed on an SSC/FSC color density plot, which was subgated onto an antigen plot for identification of CD45−7-AAD− (live) cells (gate R3). CD45−7-AAD− cells were further subgated to identify CD73+CD90+ cells (gate R4), which were then displayed as a quadrant gate to identify CD105+CD44− subset of cells (gate R5). The sort window of CD105+CD44− cells is indicated as R5. (B): Representative flow cytometric density color plots showing the response of in vitro culture-expanded elutriated/FACS-sorted CD44− cells (top), which gain CD44 expression, versus conventionally isolated mesenchymal stem/stromal cells (MSCs) (bottom) following treatment with 10 ng/ml recombinant human (rh) G-CSF over 3 days. Treatment of in vitro culture-expanded CCE/FACS CD44− cells with rhG-CSF resulted in the appearance of a CD44lo subpopulation, whereas MSCs isolated using the conventional method of plastic adherence did not. (C): Change in mean fluorescence intensity is depicted as mean ± SEM, n = 3 independent experiments. *, p < .05 versus vehicle-treated cells using an unpaired t test. Abbreviations: 7-AAD, 7-aminoactinomycin D; CCE, counterflow centrifugal elutriation; FACS, fluorescence-activated cell sorting; FSC, forward scatter; G-CSF, granulocyte-colony stimulating factor; ns, not significant; SSC, side scatter.

Figure 7.

Figure 7.

Trilineage differentiation potential of small CD45−CD73+CD90+CD105+CD44− cells obtained from fraction 90 following expansion. (A): In vitro growth of CCE/FACS CD44− cells obtained from fraction 90. Shown are a representative Giemsa-stained colony forming unit-fibroblast colony at day 12 (left) and the typical colony appearance by phase contrast (middle). After three passages, CCE/FACS-sorted cells demonstrated a greater expansion capacity compared with conventional mesenchymal stem/stromal cells (MSCs) (right). (B–D): After passage 3, cells were placed in differentiation conditions in vitro to induce osteogenesis, chondrogenesis, and adipogenesis. (B): To induce chondrogenesis, cell pellets were incubated with 10 ng/ml recombinant human transforming growth factor-β3. Chondrogenesis was detected following staining with safranin-O. A representative image of a CCE/FACS CD44− pellet (left) shows more intense staining compared with donor-matched conventional MSCs (right). (C): Osteogenesis was detected by staining cultures with 2% alizarin red S solution (pH 4.2). Shown is a representative image comparing osteogenesis cultures for CD44− cells (left) with cultures of donor-matched conventional MSCs (middle). The monolayer of CCE/FACS CD44− cells stained more densely with alizarin red S. (D): Adipogenesis was detected following staining of cultures with Oil Red O, which detects lipids. Representative images of CCE/FACS CD44− culture (left) compared with donor-matched conventional MSCs (middle) are shown (×20). Right panel shows spectrophotometric quantification of adipogenesis following isopropanol extraction of Oil Red O. Data are presented as mean ± SEM, n = 4–5. *, p < .05 versus conventional isolated MSCs, using an unpaired t test. Abbreviations: CCE, counterflow centrifugal elutriation; FACS, fluorescence-activated cell sorting.

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