Isolation of a Highly Clonogenic and Multipotential Subfraction of Adult Stem Cells from Bone Marrow Stroma
http://www.100md.com
《干细胞学杂志》
Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, Louisiana, USA
Key Words. MSCs ? Mineralizing cells ? Adipocytes ? RS cells
Correspondence: Darwin J. Prockop, M.D., Ph.D., Center for Gene Therapy (SL-99), Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112 USA. Telephone: 504-988-7755; Fax: 504-988-7710; e-mail: dprocko@tulane.edu
ABSTRACT
One possible strategy for therapy with stem cells is to use the adult stem cells from bone marrow stroma referred to as mesenchymal stem cells or marrow stromal cells (MSCs) . Human MSCs are relatively easy to obtain from a small aspirate of bone marrow under conditions in which they retain the potential to differentiate into multiple cell lineages that include osteoblasts, adipocytes, chondrocytes, myoblasts, and early progenitors of neural cells. MSCs proliferate rapidly and largely retain their multipotentiality for differentiation after expansion in culture . However, cultures of expanded cells are heterogeneous in morphology, and they lose multipotentiality as they are replated for six or seven passages . The cells are also highly sensitive to plating density, and early progenitors are rapidly lost if the cultures are grown to confluency . Additionally, there is considerable variation in the proportion of early progenitors recovered from different samples of bone marrow, even when the samples are obtained from the same donor at the same time . Currently, there are no surface epitopes that are useful for distinguishing early progenitors from mature cells in the cultures . For these reasons, it is obviously important to devise standardized protocols for isolating and characterizing MSCs.
We previously extended earlier observations and identified a subfraction of small and rapidly self-renewing cells (RS cells) in early-passage, low-density cultures of human MSCs . The RS cells were characterized by low forward scatter (FSlo) and low side scatter (SSlo) of light. In the experiments described here we demonstrate that FSlo/SSlo MSCs can be isolated by fluorescence-activated cell sorter (FACS) analysis and that the isolated cells are up to 90% clonogenic. Essentially all of the FSlo/SSlo cells differentiate into either osteoblasts or adipocytes. We also demonstrate that a rapid, standardized assay for FS/SS may be useful to identify preparations of MSCs enriched for RS cells that will expand rapidly during subsequent passage in culture. The use of the assay should help to resolve discrepancies in data obtained by different laboratories with apparently similar preparations of MSCs.
METHODS
In initial experiments, we used late-passage MSCs to further test whether MSCs can be subfractionated by FS and SS (Fig. 1). The uncorrected plot of FS versus SS indicated three distributions of events (Fig. 1A). Staining with Annexin V-FITC demonstrated that the events in the upper left of the plot were cell debris and dead cells (R1 in Fig. 1B). To obtain subfractions of cells, the Annexin V+ events were gated out, and four subpopulations were defined on the basis of FS and SS (Fig. 1C). Cells defined by their light-scattering characteristics—for example, FSlo/SSlo and FShi/SShi (R2 and R3 in Fig. 1E and the four regions in Fig. 1C)—were isolated by FACS. Reassay of the two subfractions from Figure 1E demonstrated that they were distinct in terms of their light-scattering properties and the separations were reproducible (Fig. 1D and 1F). Subsequent experiments demonstrated that the proportion of Annexin V+ events was only about 1% in early-passage, low-density cultures that were harvested by timed digestion with trypsin/EDTA. Therefore, gating to exclude Annexin V+ cells was unnecessary for early-passage, low-density cultures.
To define the properties of the subfractions, cells from each of the separate regions indicated in Figure 1C were sorted into 96-well plates at 1 cell per well (three separate experiments). The sorted cells were then incubated to develop an assay for sc-CFUs (Fig. 2C) and clonal differentiation into adipocytes (not shown) and osteoblasts (Fig. 2B). Cells that were FSlo/SSlo gave a significantly higher value in the sc-CFU assay than cells from any of the other three regions (Fig. 2C). One-way ANOVA followed by Newman-Keuls multiple comparisons was used to test for the significance of the difference in clonogenicity observed for the four regions. The difference was significant with p < .05. The clonogenicity values for FSlo/SSlo cells from different preparations ranged from 80%–90%. The assays of parallel 96-well plates incubated in either osteogenic or adipogenic medium indicated that essentially all the colonies generated from FSlo/SSlo cells differentiated into mineralizing cells (Figs. 2B, C) or adipocytes (not shown). As reported elsewhere , single cell–derived colonies obtained by low-density plating of the cells were previously shown to differentiate into mineralizing cells, adipocytes, and chondro-cytes. Therefore, the FSlo/SSlo cells correspond to the sub-fraction previously referred to as RS cells .
Figure 2. Clonogenicity and differentiation of marrow stromal cells sorted by FS/SS. (A): Single-cell colony-forming unit assays of cells sorted by FS/SS (regions FSlo/SSlo, FShi/SShi, FSlo/SShi and FShi/SSlo in Fig. 1C). Values indicate the percentage of wells that contained a colony of at least 1-mm diameter 12–14 days after sorting a single cell into each well. Error bars are standard deviations over three separate experiments, with regions sorted into parallel plates for colony differentiation. *p < .05. (B, C): Representative parallel microtiter plates in which single FSlo/SSlo cells were deposited. The plate shown in (C) was stained with crystal violet to count colonies after incubation in standard medium for 12–14 days. The parallel plate (B) was stained with alizarin red S to stain mineral after incubation in standard medium for 7 days and then in osteogenic medium for 21 days. Microscopy (4x phase) of the plates (not shown) demonstrated that all colonies were positive for mineralizing cells, but the extent of mineralization varied among colonies. Abbreviations: FS, forward scatter; SS, side scatter.
To confirm that the sc-CFU assay accurately measured single cell–derived colonies, genotyping experiments were carried out with SNPs and heteroduplex analyses of PCR products spanning the SNPs . MSCs were prepared from three different donors with distinct genotypes for SNPs in either of the two genes for type I collagen (COL1A1 and COL1A2) or the gene for type II collagen (COL2A1). Pairwise mixtures containing equal numbers of cells from two distinguishable donors were used for the sc-CFU assays, and DNA from the resulting colonies was assayed. In one experiment, colonies generated by mixtures of MSCs from donors 87 and 146 were homogeneous for either the SNP in exon 9 of COL1A1 found in donor 146 or the SNP in exon 25 of COL1 A2 found in donor 87 (Fig. 3, upper panel). In a second experiment, colonies generated by mixtures of MSCs from donors 107 and 146 were homogeneous for the SNP in exon 25 of COL1A2 found in donor 107 (Fig. 3, lower panel). Therefore each of the colonies had arisen from a single donor in 13 out of 13 colonies tested.
Figure 3. Heteroduplex analysis of colonies generated by sorting 50/50 mixtures of cell suspensions from different donors. Mixed donor cell suspensions were sorted 1 cell per well into 96-well plates and cultured for 14 days. The colonies were assayed for single-nucleotide polymorphisms by heteroduplex analysis using conformation-sensitive gel electrophoresis . The pattern of heteroduplex bands is consistent with all colonies being derived from a single donor.
The sc-CFU assay was compared with the commonly used CFU assay in which cells are plated in tissue-culture dishes at low densities of about two cells per cm2 (Fig. 4). The coefficient of variation was used as a measure of reproducibility. The coefficient of variation for the sc-CFU assay was about one-third the coefficient of variation for the commonly employed assay in which the cells were plated at low density (14.6 versus 4.52; n = 4 for each experiment). A t-test for the difference in reproducibility assuming unequal variance (Excel program) indicated a p value < .02.
Figure 4. Comparison of sc-CFU assay and the commonly used, low-density CFU assay. Passage 2 marrow stromal cells were plated at densities of 50 to 1,000 cells per cm2, incubated for 10–11 days, and lifted with trypsin/EDTA before the colony assay. The sc-CFU assay was carried out as in Figure 1B by plating Annexin V-fluorescein isothiocyanate negative cells. The low-density CFU assay was carried out by plating 100 cells in a 57-cm2 dish and counting crystal violet–stained colonies with diameters greater than 1 mm after a 2-week incubation. Values are means ± standard deviation with n = 4 for determination of the percentage coefficient of variation for each procedure (p < .02). Abbreviation: CV, coefficient of variation; sc-CFU, single-cell colony-forming unit.
To demonstrate further the differences between the cells based on light-scattering properties, cells that showed the greatest difference in FS and SS were sorted from gates R3 and R5 in Figure 1C or R2 and R3 in Figure 1E, and the mRNAs were assayed with microarrays. The data were first analyzed to select the genes whose signal intensities showed the greatest difference between the two fractions. Of the 13 genes that showed the greatest difference, eight were related to cell proliferation (Table 1). Five genes that are expressed in proliferative cells were expressed at higher levels in FSlo/SSlo cells (Fig. 5A). In contrast, three genes that are expressed in less proliferative cells were expressed at lower levels in the FSlo/SSlo cell fraction (Fig. 5B).
Table 1. Identity of genes shown in Figure 5
Figure 5. Microarray assays of mRNAs of cells sorted as FSlo/SSlo and FShi/SShi by microarray, as illustrated in Figure 1E. (A): Expression of mRNAs of genes expressed at higher levels in FSlo/SSlo cells. * Indicates genes involved in cell proliferation (see Table 1). (B): Genes expressed at higher levels in FShi/SShi cells. Abbreviations: FS, forward scatter; SS, side scatter.
In additional attempts to identify subfractions of MSCs, we stained for ?-galactosidase, a marker for senescent cells . The stain was not useful because the number of ?-galactosidase+ events in late-passage cultures (passage 6) was only twice the 1.5% threshold established for early-passage cultures (passage 3) enriched for RS cells. In another series of experiments, we attempted to identify cells comparable to those defined as side population cells (SP cells) from bone marrow . The dye (Hoechst 33342) used to identify SP cells proved to be toxic to human MSCs. In samples with 106 human MSCs/ml, essentially all the cells were killed after 18 minutes at 37°C in the presence of the recommended concentration of dye (5 μg/ml) and before dye exclusion could be assessed.
To standardize the assay for FS and SS, we used a closed stream flow cytometer (Beckman-Coulter XL with ADC) and calibrated the system with microbeads of known dimensions (Fig. 6). The microbeads, ranging in size from 7–20 μ, generated a linear and reproducible standard curve for FS (Fig. 6B). To standardize the assay, we adjusted the gains and the voltages on the photomultiplier tubes so that the FS peak for the 20-μ bead was 650 and the SS peak for the 7-μ bead was 450. To compare different samples, we compared early-passage, low-density cultures with late-passage, high-density cultures in terms of the distribution of events assayed in different regions, as defined in Figures 6A and 6C. We found that the most sensitive discriminator among the samples was a flow parameter defined as log of % Annexin V– events in region G divided by % events in region T. For example, the flow parameter for low-density cultures of passage 3 cells was 1.71 (Fig. 6A) and 0.44 for high-density cultures of passage 5 cells (Fig. 6C). Therefore, the flow parameter appears to reflect the relative content of RS cells in the preparations .
Figure 6. Standardized assay for FS/SS of MSCs. (A):Assay of passage 3 MSCs from a culture initially plated at low density (100 cells per cm2) and incubated for 4 days. The cells were lifted with trypsin/EDTA and then assayed on the closed stream flow cytometer (see Methods). (B): Calibration curve obtained with microbeads to standardize measurement of FS. The calibration curve was used to locate the vertical lines in and A and C. (C):Assay of passage 5 MSCs from a culture initially plated at high density (1,000 cells per cm2) and incubated for 4 days. Arrows indicate FS for 20-μ bead set at 650 and SS for the 7-μ bead set at 450. (D): SS calibration indicating the two peaks in SS corresponding to the 7- and 10-μ beads that were used to place the horizontal lines in (A, C).Abbreviations: FS, forward scatter; MSC, marrow stromal cell; SS, side scatter.
To assess the predictive value of the FS/SS assay, we compared 19 different preparations of human MSCs that had been isolated and expanded with slightly different protocols over a 2-year period and then stored frozen (Fig.7). The thawed vials of cells were plated at high density for 1 day to recover viable cells (P2), and then separate aliquots were assayed for FS/SS and for expansion over 4 days after plating at 100 cells/cm2 (harvested cells are P3). The data indicated that all 10 of the preparations with a flow parameter greater than 0.5 expanded fivefold or greater in 4 days. In contrast, 6 preparations with a flow parameter of less than 0.5 expanded less than fivefold. Three preparations appeared to be outliers in the assay, because they had flow parameters less than 0.5 but expanded more than fivefold. The outliers were not explained by age differences of the donors (20, 27, and 35 years for the outliers versus mean of 25.8 years ± 4.7 S.D. for all 19 donors) or gender (two males and one female for the outliers versus a 2.6 ratio of males to females for the remaining donors). The outliers were also not explained in any obvious manner by differences in the conditions used to prepare or expand the MSCs.
Figure 7. Prediction of fold change in expansion of MSCs by measuring flow parameter. Plot of flow parameter of MSCs with the observed fold expansion of same preparations plated at 100 cells per cm2 and incubated for 4 days. Frozen vials of passage 1 cells were thawed, and all the cells were incubated on a 148-cm2 plate for 1 day to recover viable passage 2 cells. The cells were lifted with trypsin/EDTA, and then separate aliquots were taken for the standardized forward and side scatter assay and expansion. The vertical dashed line separates samples with flow parameter of 0.5 or greater that showed fivefold or greater expansion on subsequent plating. The horizontal dashed line separates cultures that expanded fivefold or more. Abbreviations: MSC, marrow stromal cell.
DISCUSSION
Jason R. Smith and Radhika Pochampally contributed equally to this work.
REFERENCES
Friedenstein AJ. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 1987;20:263–272.
Owen ME, Friedenstein AJ. Stromal stem cells: marrow-derived osteogenic precursors. In: Evered D, Harnett S, eds. Cell and Molecular Biology of Vertebrate Hard Tissues. Ciba Foundation Symposium. Wiley: Chichester, U.K. 1988;136:42–60.
Mets T, Verdonk G. In vitro aging of human bone marrow-derived stromal cells. Mech Ageing Dev 1981;16:81–89.
Piersma AH, Brockbank KGM, Ploemacher RE et al. Characterization of fibroblastic stromal cells from murine bone marrow. Exp Hematol 1985;13:237–243.
Castro-Malaspina H, Gay RE, Resnick G et al. Characterization of human bone marrow fibroblast colony-forming cells (CFU-F) and their progeny. Blood 1980;56:289–301.
Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9:641–650.
Prockop DJ. Marrow stromal cells as stem cells for non-hematopoietic tissues. Science 1997;276:71–74.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Colter DC, Class R, DiGirolamo CM et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci U S A 2000;96:7294–7299.
Colter DC, Sekiya I, Prockop DJ. Identification of a sub-population of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci U S A 2001;98:8741–8745.
Sekiya I, Larson BL, Smith JR et al. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yield of early progenitors and for evaluating their quality. STEM CELLS 2002;20:430–441.
DiGirolamo CM, Stokes D, Colter D et al. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haem 1999;107:275–281
Korkko J, Annunen S, Pihlajamaa T et al. Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: comparison with denaturing gradient gel electrophoresis and nucleotide sequencing. Proc Natl Sci U S A 1998;95:1681–1685..
Ganguly A, Rock MJ, Prockop DJ. Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments. Proc Natl Sci U S A 1993;90:10325–10329.
Coates PJ. Markers of senescence? J Pathol 2002;96:371–373.
Goodell MA, Rosenzweig M, Kim H et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 1997;3:1337–1345.
McKinney-Freeman SL, Jackson KA, Camargo FD et al. Muscle-derived hematopoietic stem cells are hematopoietic in origin. Proc Natl Acad Sci U S A 2002;99:341–346.
Gregory CA, Singh H, Perry AS et al. The Wnt signaling inhibitor Dkk-1 is required for re-entry into the cell cycle of human adult stem cells from bone marrow (hMSCs). J Biol Chem 2003;278:28067–28078.
Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci 2000;113:1161–1166.
Wagers AJ, Christensen JL, Weissman IL. Cell fate determination from stem cells. Gene Ther 2002;9:606–612.
Ghilzon R, McCulloch CA, Zohar R. Stromal mesenchymal progenitor cells. Leuk Lymphoma 1999;32:211–221.(Jason R. Smith, Radhika P)
Key Words. MSCs ? Mineralizing cells ? Adipocytes ? RS cells
Correspondence: Darwin J. Prockop, M.D., Ph.D., Center for Gene Therapy (SL-99), Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112 USA. Telephone: 504-988-7755; Fax: 504-988-7710; e-mail: dprocko@tulane.edu
ABSTRACT
One possible strategy for therapy with stem cells is to use the adult stem cells from bone marrow stroma referred to as mesenchymal stem cells or marrow stromal cells (MSCs) . Human MSCs are relatively easy to obtain from a small aspirate of bone marrow under conditions in which they retain the potential to differentiate into multiple cell lineages that include osteoblasts, adipocytes, chondrocytes, myoblasts, and early progenitors of neural cells. MSCs proliferate rapidly and largely retain their multipotentiality for differentiation after expansion in culture . However, cultures of expanded cells are heterogeneous in morphology, and they lose multipotentiality as they are replated for six or seven passages . The cells are also highly sensitive to plating density, and early progenitors are rapidly lost if the cultures are grown to confluency . Additionally, there is considerable variation in the proportion of early progenitors recovered from different samples of bone marrow, even when the samples are obtained from the same donor at the same time . Currently, there are no surface epitopes that are useful for distinguishing early progenitors from mature cells in the cultures . For these reasons, it is obviously important to devise standardized protocols for isolating and characterizing MSCs.
We previously extended earlier observations and identified a subfraction of small and rapidly self-renewing cells (RS cells) in early-passage, low-density cultures of human MSCs . The RS cells were characterized by low forward scatter (FSlo) and low side scatter (SSlo) of light. In the experiments described here we demonstrate that FSlo/SSlo MSCs can be isolated by fluorescence-activated cell sorter (FACS) analysis and that the isolated cells are up to 90% clonogenic. Essentially all of the FSlo/SSlo cells differentiate into either osteoblasts or adipocytes. We also demonstrate that a rapid, standardized assay for FS/SS may be useful to identify preparations of MSCs enriched for RS cells that will expand rapidly during subsequent passage in culture. The use of the assay should help to resolve discrepancies in data obtained by different laboratories with apparently similar preparations of MSCs.
METHODS
In initial experiments, we used late-passage MSCs to further test whether MSCs can be subfractionated by FS and SS (Fig. 1). The uncorrected plot of FS versus SS indicated three distributions of events (Fig. 1A). Staining with Annexin V-FITC demonstrated that the events in the upper left of the plot were cell debris and dead cells (R1 in Fig. 1B). To obtain subfractions of cells, the Annexin V+ events were gated out, and four subpopulations were defined on the basis of FS and SS (Fig. 1C). Cells defined by their light-scattering characteristics—for example, FSlo/SSlo and FShi/SShi (R2 and R3 in Fig. 1E and the four regions in Fig. 1C)—were isolated by FACS. Reassay of the two subfractions from Figure 1E demonstrated that they were distinct in terms of their light-scattering properties and the separations were reproducible (Fig. 1D and 1F). Subsequent experiments demonstrated that the proportion of Annexin V+ events was only about 1% in early-passage, low-density cultures that were harvested by timed digestion with trypsin/EDTA. Therefore, gating to exclude Annexin V+ cells was unnecessary for early-passage, low-density cultures.
To define the properties of the subfractions, cells from each of the separate regions indicated in Figure 1C were sorted into 96-well plates at 1 cell per well (three separate experiments). The sorted cells were then incubated to develop an assay for sc-CFUs (Fig. 2C) and clonal differentiation into adipocytes (not shown) and osteoblasts (Fig. 2B). Cells that were FSlo/SSlo gave a significantly higher value in the sc-CFU assay than cells from any of the other three regions (Fig. 2C). One-way ANOVA followed by Newman-Keuls multiple comparisons was used to test for the significance of the difference in clonogenicity observed for the four regions. The difference was significant with p < .05. The clonogenicity values for FSlo/SSlo cells from different preparations ranged from 80%–90%. The assays of parallel 96-well plates incubated in either osteogenic or adipogenic medium indicated that essentially all the colonies generated from FSlo/SSlo cells differentiated into mineralizing cells (Figs. 2B, C) or adipocytes (not shown). As reported elsewhere , single cell–derived colonies obtained by low-density plating of the cells were previously shown to differentiate into mineralizing cells, adipocytes, and chondro-cytes. Therefore, the FSlo/SSlo cells correspond to the sub-fraction previously referred to as RS cells .
Figure 2. Clonogenicity and differentiation of marrow stromal cells sorted by FS/SS. (A): Single-cell colony-forming unit assays of cells sorted by FS/SS (regions FSlo/SSlo, FShi/SShi, FSlo/SShi and FShi/SSlo in Fig. 1C). Values indicate the percentage of wells that contained a colony of at least 1-mm diameter 12–14 days after sorting a single cell into each well. Error bars are standard deviations over three separate experiments, with regions sorted into parallel plates for colony differentiation. *p < .05. (B, C): Representative parallel microtiter plates in which single FSlo/SSlo cells were deposited. The plate shown in (C) was stained with crystal violet to count colonies after incubation in standard medium for 12–14 days. The parallel plate (B) was stained with alizarin red S to stain mineral after incubation in standard medium for 7 days and then in osteogenic medium for 21 days. Microscopy (4x phase) of the plates (not shown) demonstrated that all colonies were positive for mineralizing cells, but the extent of mineralization varied among colonies. Abbreviations: FS, forward scatter; SS, side scatter.
To confirm that the sc-CFU assay accurately measured single cell–derived colonies, genotyping experiments were carried out with SNPs and heteroduplex analyses of PCR products spanning the SNPs . MSCs were prepared from three different donors with distinct genotypes for SNPs in either of the two genes for type I collagen (COL1A1 and COL1A2) or the gene for type II collagen (COL2A1). Pairwise mixtures containing equal numbers of cells from two distinguishable donors were used for the sc-CFU assays, and DNA from the resulting colonies was assayed. In one experiment, colonies generated by mixtures of MSCs from donors 87 and 146 were homogeneous for either the SNP in exon 9 of COL1A1 found in donor 146 or the SNP in exon 25 of COL1 A2 found in donor 87 (Fig. 3, upper panel). In a second experiment, colonies generated by mixtures of MSCs from donors 107 and 146 were homogeneous for the SNP in exon 25 of COL1A2 found in donor 107 (Fig. 3, lower panel). Therefore each of the colonies had arisen from a single donor in 13 out of 13 colonies tested.
Figure 3. Heteroduplex analysis of colonies generated by sorting 50/50 mixtures of cell suspensions from different donors. Mixed donor cell suspensions were sorted 1 cell per well into 96-well plates and cultured for 14 days. The colonies were assayed for single-nucleotide polymorphisms by heteroduplex analysis using conformation-sensitive gel electrophoresis . The pattern of heteroduplex bands is consistent with all colonies being derived from a single donor.
The sc-CFU assay was compared with the commonly used CFU assay in which cells are plated in tissue-culture dishes at low densities of about two cells per cm2 (Fig. 4). The coefficient of variation was used as a measure of reproducibility. The coefficient of variation for the sc-CFU assay was about one-third the coefficient of variation for the commonly employed assay in which the cells were plated at low density (14.6 versus 4.52; n = 4 for each experiment). A t-test for the difference in reproducibility assuming unequal variance (Excel program) indicated a p value < .02.
Figure 4. Comparison of sc-CFU assay and the commonly used, low-density CFU assay. Passage 2 marrow stromal cells were plated at densities of 50 to 1,000 cells per cm2, incubated for 10–11 days, and lifted with trypsin/EDTA before the colony assay. The sc-CFU assay was carried out as in Figure 1B by plating Annexin V-fluorescein isothiocyanate negative cells. The low-density CFU assay was carried out by plating 100 cells in a 57-cm2 dish and counting crystal violet–stained colonies with diameters greater than 1 mm after a 2-week incubation. Values are means ± standard deviation with n = 4 for determination of the percentage coefficient of variation for each procedure (p < .02). Abbreviation: CV, coefficient of variation; sc-CFU, single-cell colony-forming unit.
To demonstrate further the differences between the cells based on light-scattering properties, cells that showed the greatest difference in FS and SS were sorted from gates R3 and R5 in Figure 1C or R2 and R3 in Figure 1E, and the mRNAs were assayed with microarrays. The data were first analyzed to select the genes whose signal intensities showed the greatest difference between the two fractions. Of the 13 genes that showed the greatest difference, eight were related to cell proliferation (Table 1). Five genes that are expressed in proliferative cells were expressed at higher levels in FSlo/SSlo cells (Fig. 5A). In contrast, three genes that are expressed in less proliferative cells were expressed at lower levels in the FSlo/SSlo cell fraction (Fig. 5B).
Table 1. Identity of genes shown in Figure 5
Figure 5. Microarray assays of mRNAs of cells sorted as FSlo/SSlo and FShi/SShi by microarray, as illustrated in Figure 1E. (A): Expression of mRNAs of genes expressed at higher levels in FSlo/SSlo cells. * Indicates genes involved in cell proliferation (see Table 1). (B): Genes expressed at higher levels in FShi/SShi cells. Abbreviations: FS, forward scatter; SS, side scatter.
In additional attempts to identify subfractions of MSCs, we stained for ?-galactosidase, a marker for senescent cells . The stain was not useful because the number of ?-galactosidase+ events in late-passage cultures (passage 6) was only twice the 1.5% threshold established for early-passage cultures (passage 3) enriched for RS cells. In another series of experiments, we attempted to identify cells comparable to those defined as side population cells (SP cells) from bone marrow . The dye (Hoechst 33342) used to identify SP cells proved to be toxic to human MSCs. In samples with 106 human MSCs/ml, essentially all the cells were killed after 18 minutes at 37°C in the presence of the recommended concentration of dye (5 μg/ml) and before dye exclusion could be assessed.
To standardize the assay for FS and SS, we used a closed stream flow cytometer (Beckman-Coulter XL with ADC) and calibrated the system with microbeads of known dimensions (Fig. 6). The microbeads, ranging in size from 7–20 μ, generated a linear and reproducible standard curve for FS (Fig. 6B). To standardize the assay, we adjusted the gains and the voltages on the photomultiplier tubes so that the FS peak for the 20-μ bead was 650 and the SS peak for the 7-μ bead was 450. To compare different samples, we compared early-passage, low-density cultures with late-passage, high-density cultures in terms of the distribution of events assayed in different regions, as defined in Figures 6A and 6C. We found that the most sensitive discriminator among the samples was a flow parameter defined as log of % Annexin V– events in region G divided by % events in region T. For example, the flow parameter for low-density cultures of passage 3 cells was 1.71 (Fig. 6A) and 0.44 for high-density cultures of passage 5 cells (Fig. 6C). Therefore, the flow parameter appears to reflect the relative content of RS cells in the preparations .
Figure 6. Standardized assay for FS/SS of MSCs. (A):Assay of passage 3 MSCs from a culture initially plated at low density (100 cells per cm2) and incubated for 4 days. The cells were lifted with trypsin/EDTA and then assayed on the closed stream flow cytometer (see Methods). (B): Calibration curve obtained with microbeads to standardize measurement of FS. The calibration curve was used to locate the vertical lines in and A and C. (C):Assay of passage 5 MSCs from a culture initially plated at high density (1,000 cells per cm2) and incubated for 4 days. Arrows indicate FS for 20-μ bead set at 650 and SS for the 7-μ bead set at 450. (D): SS calibration indicating the two peaks in SS corresponding to the 7- and 10-μ beads that were used to place the horizontal lines in (A, C).Abbreviations: FS, forward scatter; MSC, marrow stromal cell; SS, side scatter.
To assess the predictive value of the FS/SS assay, we compared 19 different preparations of human MSCs that had been isolated and expanded with slightly different protocols over a 2-year period and then stored frozen (Fig.7). The thawed vials of cells were plated at high density for 1 day to recover viable cells (P2), and then separate aliquots were assayed for FS/SS and for expansion over 4 days after plating at 100 cells/cm2 (harvested cells are P3). The data indicated that all 10 of the preparations with a flow parameter greater than 0.5 expanded fivefold or greater in 4 days. In contrast, 6 preparations with a flow parameter of less than 0.5 expanded less than fivefold. Three preparations appeared to be outliers in the assay, because they had flow parameters less than 0.5 but expanded more than fivefold. The outliers were not explained by age differences of the donors (20, 27, and 35 years for the outliers versus mean of 25.8 years ± 4.7 S.D. for all 19 donors) or gender (two males and one female for the outliers versus a 2.6 ratio of males to females for the remaining donors). The outliers were also not explained in any obvious manner by differences in the conditions used to prepare or expand the MSCs.
Figure 7. Prediction of fold change in expansion of MSCs by measuring flow parameter. Plot of flow parameter of MSCs with the observed fold expansion of same preparations plated at 100 cells per cm2 and incubated for 4 days. Frozen vials of passage 1 cells were thawed, and all the cells were incubated on a 148-cm2 plate for 1 day to recover viable passage 2 cells. The cells were lifted with trypsin/EDTA, and then separate aliquots were taken for the standardized forward and side scatter assay and expansion. The vertical dashed line separates samples with flow parameter of 0.5 or greater that showed fivefold or greater expansion on subsequent plating. The horizontal dashed line separates cultures that expanded fivefold or more. Abbreviations: MSC, marrow stromal cell.
DISCUSSION
Jason R. Smith and Radhika Pochampally contributed equally to this work.
REFERENCES
Friedenstein AJ. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 1987;20:263–272.
Owen ME, Friedenstein AJ. Stromal stem cells: marrow-derived osteogenic precursors. In: Evered D, Harnett S, eds. Cell and Molecular Biology of Vertebrate Hard Tissues. Ciba Foundation Symposium. Wiley: Chichester, U.K. 1988;136:42–60.
Mets T, Verdonk G. In vitro aging of human bone marrow-derived stromal cells. Mech Ageing Dev 1981;16:81–89.
Piersma AH, Brockbank KGM, Ploemacher RE et al. Characterization of fibroblastic stromal cells from murine bone marrow. Exp Hematol 1985;13:237–243.
Castro-Malaspina H, Gay RE, Resnick G et al. Characterization of human bone marrow fibroblast colony-forming cells (CFU-F) and their progeny. Blood 1980;56:289–301.
Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9:641–650.
Prockop DJ. Marrow stromal cells as stem cells for non-hematopoietic tissues. Science 1997;276:71–74.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Colter DC, Class R, DiGirolamo CM et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci U S A 2000;96:7294–7299.
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