Characterization of Stromal Progenitor Cells Enriched by Flow Cytometry

The progenitors for cells of bone, cartilage, fat, and muscle densed nuclei and a paucity of cytoplasmic organelles. Freshly sorted suspensions of immunocytochemically are thought to be derived from mesenchymal stem cells but despite extensive study of stromal cell differentiation, neistained S cells did not express differentiation-associated markers such as type I, II, and III collagens, alkaline phosphather mesenchymal stem cells or the more committed, tissuespecific progenitors have been well-characterized. In this tase, or osteopontin. However, after attachment, S cells became immunopositive for collagens I, II, III, osteopontin, and study we used flow cytometry to isolate from fetal rat periosteum a population of small, slowly cycling cells with low also for the cell surface receptor CD44, which mediates cell attachment to hyaluronan and osteopontin. These studies cytoplasmic granularity (S cells) that display stem cell characteristics. On plating, S cells exhibited a 90% higher labelshow that viable osteogenic precursor cells with the stem cell characteristics of self-renewal, high proliferative capacing index with [H]-thymidine compared to unsorted cells and when grown in culture generated cartilage, adipocyte, ity, and multipotentiality can be enriched from heterogeneous stromal cell populations with simple flow cytometric and smooth muscle phenotypes, in addition to bone. Only the S-cell population showed extensive self-renewal of cells methods. These cells may be useful for regeneration of stromal tissues. with osteogenic potential. Electron microscopy showed that S cells have high nuclear:cytoplasmic ratios with large conq 1997 by The American Society of Hematology.

an argon ion laser (Coherent innova 70) operating at 250 mW beam days, wells were fixed overnight in neutral buffered formalin and stained with von Kossa's reagent.The fraction of wells without bone power.Gating windows were established for forward light scatter (FSC) and side scatter (SSC).Particles with an average size õ5 mm nodules (F 0 ; nonresponsive wells) was calculated for each plating density.This fraction was plotted against the number of cells plated (determined by running standard size beads) were excluded.Two major subpopulations were sorted: cells with the lowest 15% FSC per well for each one of the subpopulations.A linear regression was fitted with 95% confidence limits and correlation coefficients were and lowest 15% SSC in the population (S cells) and the cells with the highest 15% FSC and highest 15% SSC (L cells).Three other calculated.Application of the Poisson distribution 28 permitted estimation of the number of osteoprogenitor cells in each of the groups populations were also collected: (1) FRCC cells were passed without sorting through the flow cytometer as a control group to evaluate which upon division was capable of forming bone nodules.Colony assays.For assessment of osteogenic capacity at rela-the effect of passage through the flow cytometer on osteogenesis in vitro and other functional assays; (2) cells remaining after sorting tively high cell densities, sorted cells were plated in 35-mm dishes at 3 1 10 4 cells per dish.Two sets of dishes were prepared, either the two main groups (S, L), were designated as S 0 and L 0 , respectively.All groups were collected in glass tubes containing 3 mL a- untreated (plastic) or precoated with a thin film of collagen type I (Vitrogen; Celtrix, Santa Clara, Ca) following the supplier's proto-MEM with 15% FBS and 10% antibiotics.
Cell vitality.To assess cell vitality as a result of passage through col.Cultures were subsequently augmented with 50 mg/mL ascorbic acid and 10 mmol/L sodium b-glycerophosphate (Sigma) at conflu-the flow cytometer and sorting, sorted cells were plated overnight into 8-well chamber slides (n Å 8 replicates; Nunc, Roskilde, Den-ence.The cultures were terminated on day 16, fixed overnight in neutral buffered formalin, and stained with von Kossa's reagent. 29mark) at a density of 1 1 10 3 cells per well.One slide was plated at the same density with unsorted FRCC.Slides were washed with The number of bone nodules was counted in each plastic dish.Triplicate fields (101 objective) for each culture were counted in triplicate PBS to remove nonviable cells and debris and fixed.Cells were washed, permeabilized with 0.01% Triton-X (BDH, Toronto, ON)  dishes.The mean number of bone nodules and standard deviations were calculated for each group.The mean area of bone nodule was to permit intracellular staining and incubated for 10 minutes at 4ЊC with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; Boeh-assessed by image analysis (R&M Biometrics, Nashville, TN) and expressed as mm 2 ; 30 replicate nodules were measured for each ringer, Mannheim, Germany; 1 mg/mL final concentration in 0.1% NP-40).Slides were analyzed by immunofluorescence microscopy.
group (101 objective).The mean nodule size and standard deviations were calculated and differences were evaluated using analysis of The mean number of cells and standard errors of the means per microscopic field area (251 objective) were computed.Statistical variance.Collagen-coated dishes exhibited diffuse patterns of mineralization that precluded enumeration of bone nodules.analysis of computation of P values for these data were determined using analysis of variance and Tukey's test.
Self-renewal capacity.To assess the self-renewal capacity of cells from the two main sorted subpopulations (S, L), and FRCC OPN mRNA.Cells were analyzed for the presence of OPN mRNA by the reverse transcriptase polymerase chain reaction (RT-passed through the flow cytometer without sorting, cells were plated in T-25 flasks at a plating density of 7.5 1 10 4 cells per flasks and PCR) as described. 22RNA was extracted using the Mini-GT Protocol for preparation of total nucleic acids. 26The GeneAmp RNA PCR grown for 12 days (early mineralization stage 22 ).Nonviable cells and debris were removed by washing twice with PBS and attached Kit (Perkin-Elmer Cetus, Norwalk, CT) was used for cDNA synthesis and PCR amplification following the supplier's protocol with cells were procured with trypsin in citrate buffer.Visual inspection of cultures following trypsinization confirmed the complete removal minor modifications.Random hexamers were used to prime the cDNA synthesis (20 mL final volume) from the RNA of 200 cells of all cells.Aliquots were counted electronically and prepared for sorting as described above.Initially, cells were resorted into 3 sub-as described. 22Seminested PCR was performed to confirm that the amplified product was derived from OPN cDNA.Amplification populations (parental FRCC s ; S s ,L s ) using the same criteria as the first sort.Subsequent sorts used expanded windows for the L subpop-products were analyzed by electrophoresis in a 2% agarose gel in 0.51 Tris borate EDTA (TBE) and visualized with 0.5 mg/mL ethid-ulation.Cells were seeded into 12-well chamber plates at a plating density of 1.5 1 10 4 cells per well and supplemented with ascorbic ium bromide.
Alkaline phosphatase activity and cell proliferation.Sorted cells acid (50 mg/mL) and 10 mmol/L sodium b-glycerophosphate at confluence.Cultures were terminated after 16 days of culture, stained were plated into 8-well chamber slides (n Å 4 replicates; Nunc) at a plating density of 1 1 10 3 cells per well and were compared to with von Kossa's reagent and analyzed for bone nodule formation.The mean and standard deviation of the number of bone nodules unsorted FRCC plated at the same density.Cultures were analyzed on day 1, 2, 4, 6, 8, and 10.Due to the limited numbers of cells in were determined from duplicate fields (101 objective) counted for each culture in 6 replicate dishes.the sorted subpopulations, only FRCC were analyzed on day 12.To assess the proportion of proliferating cells, wells were incubated Electron microscopy.Transmission electron microscopy was performed to assess the morphological characteristics of the subpop-with 3 H-thymidine (1 mCi/mL) for 3 hours before the termination of the culture.Slides were fixed, stained for alkaline phosphatase ulations sorted by flow cytometry.Sorted subpopulations were either pelleted or plated overnight, and then fixed with 2.5% glutaraldehyde (AP) activity, and prepared for radioautography with NTB-2 liquid emulsion (Kodak, Rochester, NY) as described. 27The following in 0.1 mol/L sodium cacodylate buffer, pH 7.3, postfixed with osmium tetroxide, dehydrated in an ethanol series, and embedded in populations of cells were counted: 3 H-thymidine labeled cells (ú4 silver grains per nucleus), AP / cells, and cells with both 3 H-thymi-spurr epoxy resin.Thin sections (7 nm) were stained with uranyl acetate and lead citrate and examined under an electron microscope.dine labeling and AP staining.All cells were counted in the same microscopic field area (401 objective; triplicate fields for each cul-Immunostaining.The two sorted subpopulations (S and L) were characterized by antibody staining and the staining patterns were ture).The mean labeling indices of cells and standard errors of the means were computed.
compared with unsorted FRCC.Two sets of staining procedures were used.In the first procedure, 1.5 1 10 4 cells were sorted by Limiting dilution analysis.Cells were plated in 96-well plates at dilutions of 5, 25, 50, 100, 300, and 600 cells per well.Two flow cytometry, collected in tubes, fixed immediately, stained in suspension, and cytospin preparations prepared as described. 10In the replicate plates (192 wells) were obtained for each dilution.Cells were grown continuously for 24 days and the medium was changed second procedure, sorted cells were plated in 8-well chamber slides (Nunc) at a plating density of 2  FITC / cells divided by the number of DAPI nuclei.

RESULTS
ulation was the highest of all the subpopulations in the first 48 hours of culture.In the first 24 hours of culture the S We assessed whether FRCC could support hematopoiesis, thereby indicating their potential as stromal cells.Nonadher-subpopulation exhibited a 90% higher labeling index than the unsorted FRCC and ú100% more than the L subpopula-ent hematopoietic cells from either rat or mouse femoral BM flushes did not form colonies when plated in methylcellulose, tion.S-cell nuclei were heavily labeled with silver grains (ú20 grains/nucleus) whereas the other subpopulations ex-IMDM, and 2% FBS.However, when incubated with growth factors (3 U/mL erythropoietin, 10 ng/mL mrIL-3, 10 ng/ hibited only 5 to 12 grains for labeled cells.The two sorted subpopulations (S, L) showed decreased proliferation during mL hrIL-6, and 50 ng/mL mr Stem Cell Factor; all from Stem Cell Technologies), colony-forming units (CFC-C) the matrix formation phase of the culture (days 4 through 6) while FRCC maintained a relatively constant % of labeled were observed (CFC-C 049 { 3.9/35-mm dish).Similarly, when cells were incubated with IMDM and 2% FBS and cells.As cultures multilayered and approached mineralization (days 8 through 12), there was another increase in the plated on feeder cell layers of either high density rat BM stromal cells (CFC-C 057 { 4.6/35-mm dish) or on high percentage of labeled cells for all groups.Although at day 8 there was no difference between the subpopulations, the density FRCC (CFC-C 054 { 3.45/35-mm dish), there was colony development and with no statistically different colony S subpopulation at day 10 exhibited threefold more labeled cells than the other two groups.counts compared to the number of colonies in cultures sup-AP activity (Fig 2B ), used as a marker of osteoblastic differentiation, was detected in Ç35% of cells in the FRCC and L groups during the first 24 hours of culture whereas S cells did not show any detectable AP activity over this time period.All groups showed increased proportions of AP / cells during the matrix formation phase in which the highest proportion were in the S and L groups (70% of the cells) and 60% of the cells in the FRCC.The L cells exhibited high proportions of AP / cells until day 8 when there was a decrease to only 30% of the cells.A similar decline was observed in the FRCC on day 10 and these two groups showed increased proportions of AP cells by 5% to 15% as they approached mineralization.The S population did not lose the high proportion of AP / cells acquired during culture: at least 60% of the cells on day 6 were AP / .AID Blood 0048 / 5h40$$$941 10-10-97 16:42:07 blda WBS: Blood The same fields were analyzed for cells exhibiting dual labeling for [ 3 H]-thymidine and AP, a measure of the proportion of transit amplifying cells.In the first 4 days only cultures of the parental FRCC and L cells showed dual-labeled cells (Fig 2C).The peak proportion for the L population was on day 2 when 40% of the cells in the culture were double labeled whereas only 23% were double labeled for the FRCC.The S group did not show dual-labeled cells until day 6 when all groups exhibited similar proportions of duallabeled cells (20% to 30% of the cells).All groups showed an increase in the proportion of dual-labeled cells as they approached mineralization: 60% of the cells in the S group compared to 15% to 20% in the FRCC and L groups were double labeled.
Analysis of osteoprogenitor cells.Limiting dilution analysis was used to assess cell cooperativity in bone nodule formation and the relative proportions of transit amplifying cells that could divide in culture and produce bone nodules. 18he analyses showed a linear relationship between the number of cells plated per well and the fraction of nonresponsive wells.The lowest R 2 value for all groups was 0.83 in the FRCC (Fig 3A ), showing a strong linear correlation and single hit kinetics, as evaluated by counting of von Kossa stained bone nodules.Thus, the progenitor cells that divided to form nodules did not require assistance from other cell types.
Based on the single event theory 28 the proportion of amplifying osteoprogenitors was estimated for each of the subpopulations.By calculating the probability of no response at F 0 Å 0.37, we estimated that the proportion of these progenitors in the FRCC was 1:620 cells, threefold less than the S subpopulation that we estimated as 1:202 (Fig 3B ) or Ç1:120 when adjusted for cell viability (Fig 1B).The L group showed threefold lower numbers in relation to FRCC (Fig 3C ; 1:1750) that was almost ninefold less than S cells.The S 0 subpopulation contained more progenitors than L cells (Fig 3D ; 1:968), but this was still fourfold to fivefold less than the S subpopulation.Thus, over the time period of culture, all populations were capable of generating transit amplifying cells for the osteogenic lineage, but the relative frequency of these cells was much higher in the S population.
Osteogenic capacity of sorted subpopulations.We plated cells on either tissue culture plastic or on collagen- blasts. 33Within 24 hours after flow cytometry, all sorted R is the correlation coefficient.F0.37 is an estimate of the mean numsubpopulations attached to plastic or collagen-coated dishes.

ber of cells that would include one progenitor cell, based on application of the Poisson distribution.
On the basis of phase contrast microscopy and staining with oil red O, alcian blue, and toluidine blue, there was the morphological appearance of fat cells, chondrocytes, and smooth muscle cells (see below) in addition to the presence collagen gels and never to the same degree as observed in of osteogenic cells that are the focus of this report.On both the S and L 0 populations.S cells grown on plastic formed collagen and plastic substrata, cell proliferation was most rapidly mineralizing bone nodules by day 9, as observed rapid in the S group.Indeed, only the S and L 0 populations by phase-contrast microscopy.In contrast, mineralized bone had reached confluence on collagen gels by day 13, at which nodules were not evident in the L 0 and other subpopulations time a diffuse pattern of mineralization was evident.In two before day 12.However, on day 13 all groups (data not of four replicate experiments the FRCC, S 0 , and L subpopushown) had produced bone nodules except the the L population which formed small nodules 3 to 4 days later.Notably, lations showed only limited mineralization after 20 days on

. Analysis of bone nodule formation. (A) Photograph of day 16 cultures stained by Von Kossa to show mineralization. All subpopulations exhibit mineralization when grown on plastic dishes (P) but S and L Ï cells exhibit larger numbers of bone nodules. Mineralization is observed only for S and L Ï cells when grown on collagen-coated (C) dishes. (B) Bone nodule number and size (mm) determined after Von Kossa staining of the day 16 cultures grown on plastic dishes (mean Ô SEM).
bone nodules formed by the S cells were significantly larger FRCC.Quantification of bone nodules on plastic dishes (Fig 4B ) showed fourfold higher numbers of nodules in the S than those formed by the other populations (see below).
Staining of day 16 cultures with Von Kossa's reagent (Fig group than the FRCC population and 10-fold larger size of individual bone nodules (P õ .001).In contrast, the number 4A) confirmed the observations by phase contrast microscopy.Analysis of bone nodule-forming capacity of different of nodules in the L group was threefold lower than the FRCC population which was similar to the S 0 subpopulation, and subpopulations showed that in collagen-coated dishes, most of the dish in S and L 0 subpopulations was covered with twofold lower than the L 0 subpopulation.There were no significant differences in the size of nodules produced by mineralized tissue.There were small foci of mineralization in the S 0 group, but no bone nodules in the L group or the parental FRCC and L cells while the nodules produced AID Blood 0048 / 5h40$$$941 10-10-97 16:42:07 blda WBS: Blood by S 0 cells were fourfold larger than FRCC and bone nodules munocytochemical analysis of cytospin preparations showed large numbers of DAPI-stained nuclei for all preparations.produced by L 0 cells were sixfold larger than FRCC (P õ .001).
S cells did not stain for OPN (Table 1).FRCC exhibited intracellular OPN staining in Ç80% of the cells and staining Self-renewal capacity.As counts of bone nodules showed that all cultures contained measureable numbers of intensity ranged from low to high; 60% of the L cells were stained for OPN at medium intensity.High-intensity colla-amplifying osteoprogenitor cells albeit at different proportions, we conducted a more rigourous assay to estimate the gen I staining was detected in 70% of the FRCC while low to medium staining was seen in õ50% of the L cells.Medium self-renewal capacity of more primitive, undifferentiated cells.After 12 days of growth, S and L cells and FRCC were intensity collagen II staining was detected in more than 30% of the FRCC and 70% of the L cells showed medium-high-individually subjected to a second round of flow cytometry to produce S s , L s and parental (unsorted) FRCC s populations intensity staining for type II collagen.None of the S cells were immunopositive for collagens I or II.Collagen III stain-of resorted cells.Notably, on passage through the flow cytometer, 43% of S 0 cells retained the same forward scatter ing was seen in only a few S and FRCC cells (õ5%) with low intensity while 20% of the L cells exhibited low to and side scatter characteristics of the initially sorted population.In contrast, 0% of the L 0 cells exhibited the cytological medium collagen-III staining.Immunoreactivity for CD44 was not observed in any of the groups.characteristics of S 0 cells.Consequently we expanded the sorting windows for the L s population to include those cells Analysis of sorted cells plated overnight showed large numbers of attached cells stained with DAPI.High intensity with the highest 50% side scatter and highest 50% forward scatter.After plating, the S 0 -cell populations from all groups intracellular OPN staining was observed in most of the plated S cells and bright staining was also observed in 70% of the were confluent by day 7 to 8. Phase contrast microscopy of S s cells showed a progressive increase in cell numbers and FRCC and the L cells (Table 1).All groups exhibited a perimembranous, focal adhesion type of OPN staining. 22foci of bone nodule formation for each sorted population.However, L s and FRCC s groups exhibited decreased cell Larger intracellular clusters of OPN staining were observed in the L cells.High intensity but diffuse intracellular staining density from day 8 onwards.L s cells showed cell degeneration, as indicated by enlargement of cell bodies, an increased for collagen I was observed in 40% of S cells and ú60% of the FRCC and L cells.High-intensity diffuse intracellular number of cytoplasmic vacuoles and detachment of cell processes.By day 14 there were few cells remaining in the L s staining for collagen II was observed in 40% of S cells and FRCC and 80% of the L cells.Bright staining for collagen group with somewhat higher cell numbers in the FRCC s .Von Kossa staining of day 16 cultures showed abundant III was observed in 25% of the S cells while 80% of the FRCC cells showed medium intensity staining and 40% of bone nodule formation in cells from the S s group, but only barely detectable numbers of bone nodules were observed the L cells showed low-medium intensity.High intensity surface-staining for CD44 was observed in 40% of S cells in the FRCC s and even lower numbers in the other subpopulations.The mean bone nodules counts per field were: and medium staining for CD44 was observed in Ç40% of FRCC and L cells.FRCC 0 s 1.1 { 1.07; L 0 s 0.0; S 0 s 54.5 { 5.02.Pluripotentiality.In S 0 -cell cultures plated at low den-DISCUSSION sity (1,000 cells per 100-mm diameter dish; 45 days growth), isolated colonies were marked and monitored over time.
Stem cell enrichment.Stromal cells derived from bone From serial morphological observation we determined that and other mesenchymal tissues comprise a heterogeneous the colonies arose from single cells (Fig 5A).Many of the population that include cells with high proliferative capacity colonies stained positively for oil red (a marker of adipoand multipotentiality, indicative of the presence of stem cytes), for alcian blue (a marker of cartilage), and for von cells. 7However, before our study, cells with the classical Kossa's reagent (a marker for osteogenesis; Fig 5B through features of stem cells have not been clearly identified in cell D).In some colonies derived from S 0 -cell cultures, stained populations derived from stromal tissues and only limited cells of a single phenotype were colocalized with cells in progress has been made in isolating these cells. 12We have the same cluster that were of other phenotypes (ie, fat/cartishown here that sorting cells on the basis of size and cytolage or bone/fat).In contrast, stained colonies were either plasmic granularity enriches for a population of slowly cyabsent or were very rare in L 0 -cell cultures.
cling cells that did not express differentiation associated Ultrastructural characterization.Electron microscopy markers and which upon plating develops high-proliferative of the sorted cells verified the flow cytometric measurement capacity, multipotentiality, and capacity for self-renewal.In of size and cytoplasmic granularity which was used for sortcontrast, the remaining S 0 and L populations were depleted ing in this study (data not shown).L cells exhibited the of stem cells as shown by their lack of self-renewal capacity, largest size and most well-developed endoplasmic reticulum their limited proliferative capacity, and their reduced ability and vacuolar apparatus while S cells were the smallest and to form bone nodules.As these populations contained abunleast developed in terms of cytoplasmic structure.Higher dant proportions of cells that were stained for alkaline phosmagnification showed that S cells had a high nuclear/cytophatase and were labeled with 3 H-thymidine, they likely plasmic ratio, condensed nucleus, and low amount of cytocomprise transit amplifying cells. 2 Some of these putative plasmic organelles.L cells showed relatively small nuclei amplifying cells entered the osteogenic lineage and were and a cytoplasm rich in organelles.therefore capable of producing bone nodules; but they exhibited almost no self-renewal capacity.Immunofluorescence of cytospins and spread cells.Im- Since recovery of viable cells following selection and iso-paucity of cell attachment receptors such as CD44 and/or lation is problematic, 12,20 we used FRCC primary cultures as the inclusion of small apoptotic cells.a model since this system contains a relatively high propor-Cell characterization.Although multiple cell lineages, tion of osteoprogenitor cells 18 and stem cells are likely to be including adipocytes, chondrocytes, and smooth muscle cells present in the periosteal tissue surrounding bone. 6Second, were generated when S cells were grown in culture, we we sorted for a slow cycling, OPN 0 , small cell population focused our studies on the more prominent, osteogenic powith low granularity and protein content, identified pretential of these cells.When grown on either plastic or collaviously in FRCC 22 after 2 days of culture, a time at which gen substrata, S cells exhibited the sequential expression of enrichment with osteoprogenitor cells was anticipated. 16,34henotypic markers associated with the progressive phases Although flow cytometry based on selection of cells for bone of osteogenesis that have been characterized in unfractionlineage markers is potentially more discriminating (eg, Long ated calvarial cell populations. 14,15Proliferation was the domet al 21 ) and can isolate cells capable of producing bone nodinant process in the first 48 hours of all cultured subpopulaules in vitro, 20 such cells are already committed to osteoblastions.As shown in double labeling experiments, ú80% of tic differentiation and lose their osteogenic capacity follow-S cells proliferated, but did not exhibit AP activity, which ing flow sorting. 20Nevertheless, despite using relatively is an early marker of osteogenic differentiation.The major atraumatic procedures for cell separation, cell viability was shifts in AP activity and proliferation of S cells over time lowest in the S subpopulation (60%), which may reflect the in culture indicate that some of these cells, which initially did not express OPN or collagen type I, have the ability to rapidly mature and differentiate, as would be expected for For personal use only.on July 15, 2017.by guest www.bloodjournal.orgFrom sorted populations, we used a limiting dilution analysis.The estimated proportion of progenitors in the S cultures (1:202; or 1:120 if corrected for cell viability) was threefold higher than the parental, FRCC population.However, as differentiasize is altered. 2 Consistent with this prediction and as distion of multiple lineages evidently occurs in the S 0 cell cussed above, we observed dramatic phenotypic changes of subpopulation, the number of stem cells in the S subpopula-S 0 cells within hours after plating.These alterations were tion is likely to be much higher than that indicated simply not observed in L 0 cells, indicating that at least in culture, by the proportion of osteogenic progenitors.
the progenitors in the L and S 0 cell populations behave very Previous studies have indicated that osteoprogenitor cells differently.in fetal rat calvarial cultures are alkaline phosphatase posi-Stromal progenitor cells.Collectively, our studies have tive and have limited self-renewal capacity, 20 characteristics characterized a relatively undifferentiated cell population dethat are consistent with the L 0 cells described in our report.
rived from fetal rat periosteum.The features of small size, However the S 0 cells also generate bone nodules albeit at a low granularity, low cytoplasmic to nuclear ratio and undemuch higher frequency and unlike L cells, are capable of tectable expression of osteopontin, collagens, and alkaline extensive self-renewal.Further, the nodules produced by the phosphatase activity are consistent with the expectations of S cells are 10-fold larger than the L cells, indicating that the a mesenchymal stem cell (Table 2).Further, on plating, these osteogenic, transit amplifying cells produced by the S cells cells proliferated rapidly and generated new self-renewing undergo at least 3 more cell divisions than the progeny of cells as well as transit amplifying cells (lineage directed L cells.Thus, when nodule formation, nodule size, and selfcells) that expressed early lineage markers, such as collagen renewal capacity are used as the criteria for osteoprogenitor I, AP, and OPN for the osteogenic lineage.Further differenticells, it is evident that there are different classes of progenitor ation of these cells was promoted by the formation of a cells which can ultimately produce bone-forming cells.Furcollagen matrix beneath the cells, consistent with the obserther, the wide phenotypic differences between the sorted L vation that tissue nodules are formed following the producand S 0 cell populations suggest the existence of very differtion of an extracellular matrix by fully differentiated cells. 13ent types of subpopulations.Notably, stem cells alter their At this stage it is conceivable that the size of the tissue nodule is also increased through the clonal expansion of behavior markedly when their environment or compartment

(
Fig 1B) showed that, in comparison to unsorted FRCC, the following diluted antibodies: an affinity purified sheep antirat passage of cells through the flow cytometer reduced FRCC collagen I (1:20) was incubated for 1 hour at 4ЊC; an affinity purified vitality only slightly (P ú .2).However, the L and L 0 subrabbit antirat collagen II (1:50) was incubated for 1 hour at 4ЊC; an populations showed Ç20% to 25% loss of vitality (P õ .05)affinity purified sheep antipig collagen III (1:20) was incubated for compared to unsorted FRCC, whereas the S subpopulation 871 base pair (bp) sequence of the translated OPN mRNA washed twice again with PBS (Ca 2/ and Mg 2/ -free) and cytospins generated a positive response in all the sorted subpopulations of the cell suspension were prepared.Slides were coverslipped with except the S cells, which were negative in four replicates Permount (Fisher, Toronto, Ontario, Canada) and analyzed by immuand weakly positive in one replicate.When the primary PCR nofluorescence microscopy.DAPI fluorescence was used to ensure product was re-amplified using nested primers corresponding the presence of cells and then cells were examined for specific to the carboxy-terminal half of OPN, the resulting 486-bp proteins with the FITC-labeled reagents.FITC fluorescence was product confirmed the presence of OPN mRNA in all the graded as: none, low, medium, and high.The relative percentage of subpopulations except S cells, which were negative in all stained cells was estimated from counts of the relative number of replicates (Fig 1C).

Fig 1 .
Fig 1. Separation of FRCC by flow cytometry.(A) A representative flow cytometry plot of FRCC separated according to light scatter characteristics reflecting cytoplasmic granularity and cell size.Seven independent replicates of the sorting procedure were used to isolate the S (lowest 15% FSC and lowest 25% SSC) and L (highest 15% FSC and highest 25% SSC) subpopulations.This cytogram shows debris close to the origin but particles smaller than 5 mm were excluded from the sort.(B) Histogram showing the number of sorted cells (; mean Ô SEM) in each population that attached to glass after overnight plating compared to replated FRCC from the same cultures ( ).Counts were done on DAPI-stained cells and expressed as number per 100 cells plated.(C) Analysis of OPN mRNA expression in sorted cells measured by RT-PCR.Lanes 1 through 5, S cells; FRCC; L cells; Fig 2. Analysis of cell proliferation and alkaline phosphatase ex-S Ï cells; L Ï cells.First, amplification of an 871-bp fragment, encompression.(A) The proportion of 3 H-thymidine-labeled cells at differpassing most of the OPN sequence, was performed using total RNA ent stages of cellular differentiation was determined (mean labeling extracted from the cells (left side).This was followed by amplification index Ô SEM) after a 3-hour pulse of 3 H-thymidine to detect proliferatof a 486-bp fragment using the 871 fragment as template (right side), ing cells.(B) The proportion of cells expressing alkaline phosphatase which confirmed OPN expression in all populations except the S (AP index) at the same time points analyzed for cellular proliferation population.was determined by staining cells with Naphthol AS Phosphate substrate containing Fast Blue BB salts and the proportion of blue stained cells was counted (mean index Ô SEM).(C) Proportion of cells exhibiting both labeling for [ 3 H]-thymidine and AP staining (mean Ô SEM).

Fig 4
Fig 4. Analysis of bone nodule formation.(A) Photograph of day 16 cultures stained by Von Kossa to show mineralization.All subpopulations exhibit mineralization when grown on plastic dishes (P) but S and L Ï cells exhibit larger numbers of bone nodules.Mineralization is observed only for S and L Ï cells when grown on collagen-coated (C) dishes.(B) Bone nodule number and size (mm) determined after Von Kossa staining of the day 16 cultures grown on plastic dishes (mean Ô SEM).

Fig 5 .
Fig 5. Evidence for pluripotentiality by formation of multiple cell types at clonal cell densities.Sorted S Ï cells were plated at very low density (1,000 cells/ 100-mm diameter plate) and cultured for 45 days (A) Ì 4 magnification.Note the discrete, isolated colonies distributed througout the dish.Colonies were marked, observed over time, and found to arise from single cells.Colonies were stained for cartilage by alcian blue (B), for adipocytes by oil red (C) and for bone by von Kossa's reagent (D).(B) through (D) Ì 400 magnification.
32lls, as OPN and hyaluronan, which is expressed early in development, are important ligands for CD44.32Cellsimmuno-thecell cycle.A typical profile of FRCCs sorted on the basis stained for collagens type I and II were fixed with methanol at 020ЊC of light scatter is shown in Fig 1A and is representative of for 15 minutes.Samples immunostained for collagen III, OPN, CD447 separate sorting experiments that were conducted.Cell were fixed with 2% paraformaldehyde in Ca 2/ and Mg 2/ -free PBS counts showed that both the S and L cell populations comfor 30 minutes at 4ЊC.Washing and dilution of all antibodies was prised 6% to 11% of the total number of fractionated cells. in 0.25% bovine serum albumin (BSA) in PBS (Ca 2/ and Mg 2/ -free) Analysis of cells that attached following overnight plating except as outlined below.After two washes cells were incubated with H-CAM; Clone OX-49; Pharmingen, San Diego, CA) was used to low protein content and enrichment in G 1 and S phases of stain

Table 1 . Phenotype of S and L Cells osteoprogenitor
cells.Notably, there appeared to be an apparently reciprocal relationship between the relative propor- tion, all subpopulations exhibited increased proliferation, innone, / low, // medium, /// high, //// very high; () indicates dicating that bone nodule-forming cells might undergo clonal percentage of cells expressing the specific phenotypic marker.DAPI expansion during bone nodule formation.In support of this staining was used to show present of viable cells in each population; concept a quiescent cell population has been shown to prolifirrelevant antibody and 2nd Ab alone was used to show the absence of nonspecific staining.erate extensively at this phase of culture, 22 while clonal AID Blood 0048 / 5h40$$$941 10-10-97 16:42:07 blda WBS: Blood