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Stem Cells, Vol. 15, No. 5, 368-377, September 1997
© 1997 AlphaMed Press

Osteoblast Precursor Cells are Found in CD34+ Cells from Human Bone Marrow

J.-L. Chen, P. Hunt, M. McElvain, T. Black, S. Kaufman, E. S.-H. Choi

Amgen Inc., Thousand Oaks, California, USA

Key Words. Human • CD34+ cells • Osteoblast precursor cells • Osteoblasts • Bone marrow • In vitro

Dr. E. S.-H. Choi, Amgen, Inc., Mailstop 1-1-A, Amgen Center, Thousand Oaks, CA 91320, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is known that osteoblast precursor cells are found in the low-density mononuclear (LDMN) fraction of human bone marrow (BM) aspirates. The purpose of this study was to investigate whether CD34, a hematopoietic progenitor cell marker, is present on osteoblast progenitor cells. LDMN, CD34+, and CD34– cells were cultured under conditions that promote growth and differentiation of mineral-secreting osteoblasts in a limiting dilution manner. With LDMN cells, osteoblast progenitor cells were found at an average frequency of 1/36,000 cells. With CD34– cells, osteoblast progenitor frequency remained at an average of 1/33,000, similar to LDMN cells. With CD34+ selected cells, osteoblast progenitor frequency increased to an average of 1/5,000. This osteoblast progenitor frequency is maintained in sorted CD34+/CD38+ cells. The osteoblasts generated from CD34+ cells were morphologically normal, and expression of skeletal-specific alkaline phosphatase and osteonectin increased upon differentiation induced by dexamethasone (DEX) treatment. Ultrastructurally, these CD34+ cell-derived osteoblasts displayed osteoblast-specific features. Functionally, these CD34+ cell-derived osteoblasts differentiated with DEX treatment, increased the level of cyclic adenosine monophosphate in response to parathyroid hormone stimulation, increased the level of alkaline phosphatase activity, and increased mineral secretion. These results demonstrate that osteoblast progenitor cells are enriched in the CD34+ cell population from BM and that these progenitor cells can differentiate into functional osteoblasts in culture.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Osteoblasts, whose specialized functions include synthesis and secretion of the bone components (extracellular matrix proteins, proteoglycans and mineral components), are commonly believed to arise from mesenchymal stem cells (MSCs) or stromal precursors [1-4]. MSCs are a relatively ill-defined population of early progenitor cells, thought to generate fibroblasts, chondrocytes, myocytes, adipocytes, and osteoblasts [5]. To date, no specific markers or characteristics are available to identify MSCs, making the study of these cells difficult. Nonetheless, the osteoblast progenitor cells have been shown to be present in human bone marrow to give rise to preosteoblasts [6-11] and the latter have been shown to differentiate into functional osteoblasts in vitro [6, 7, 11].

Fibroblast-colony forming unit (CFU-F) [12] assays traditionally have been used to quantify stromal cell precursors of the bone marrow. Simmons and Torok-Storb reported the generation of CFU-F from sorted CD34+ cells of the human bone marrow [13], demonstrating that a possible overlap may exist between the hematopoietic and stromal precursor cells. When these sorted CD34+ cells were cultured in a long-term marrow culture system, a heterogeneous population of cells including fibroblasts, adipocytes, smooth muscle cells, and macrophages were generated [13]. To investigate if a hematopoietic cell marker, CD34, is also present on osteoblast progenitor cells, CD34+ cells from human bone marrow and fetal liver were cultured under osteogenic permissive conditions. CD34+ cells from bone marrow and fetal liver gave rise to mineral-secreting osteoblasts. Due to the increased availability of human bone marrow compared to fetal liver, the rest of the studies were conducted using cells of the human bone marrow.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All tissue culture reagents were purchased from GIBCO-BRL (Gaithersburg, MD); tissue culture vessels were purchased from Falcon (Becton-Dickinson Labware; Lincoln Park, NJ); a test of various lots of fetal bovine serum (FBS) was conducted to select a lot that gave rise to mineral-forming osteoblasts (Alizarin red+) relatively rapidly (7-10 days) and that gave rise to relatively low numbers of adipocytes (Oil-O red+) and chondrocytes (Alcian blue+). The selected FBS lot (SF 50310) was purchased from Cansera, Inc. (Ontario, Canada); all other chemicals were purchased from Sigma (St. Louis, MO); and all human bone marrow low-density mononuclear cells, cord blood CD34+ cells, and fetal liver CD34+ cells were purchased from Poietic Technologies (Gaithersburg, MD), unless otherwise indicated. Peripheral blood CD34+ cells were isolated from purchased leukapheresis units (Hemacare Inc.; Van Nuys, CA) of normal healthy paid volunteers with informed consent.

Isolation of CD34+ Cells from Human Bone Marrow and Peripheral Blood Leukapheresis Units
CD34+ cells from normal human bone marrow and normal peripheral blood leukapheresis units were isolated from low-density mononuclear cells separated by Ficoll-Paque (Pharmacia Biotech; Piscataway, NJ), and further selected using Miltenyi Biotec's (Auburn, CA) CD34 isolation kits and MS columns. The purity of CD34+ cells was determined by conventional flow cytometry using conjugated CD34 antibody (HPCA-2) and isotype controls from Becton-Dickinson (San Jose, CA) and/or by immunohistochemical staining as previously described [14] (average 94.9 ± 3.0% CD34+ cells, n = 15).

Generation of Osteoblasts from CD34+ Cells: Culture Conditions
Frozen or freshly-isolated CD34+ cells were plated at a density of 5 x 105 cells/ml, in 24-well plates (500 ul/well) in {alpha}-minimal essential medium [{alpha}-MEM] with 10% FBS (pre-selected, see above) and 1X P/S/glutamine. This medium is referred to as osteoblast growth medium or ObGM. The medium was not changed for the first seven days, but was changed two to three times per week thereafter. Once the cells reached about 90% confluence, trypsin-EDTA was used to subculture the cells into either 24-well plates or 96-well plates at a density of 4 x 104 cells/ml, (500 ul/24-well or 100 ul/96-well, respectively). When the secondary culture vessel was approximately 70%-80% confluent with preosteoblasts, ObGM was supplemented with 10 mM ß-glycerophosphate, 50 ug/ml ascorbic acid. This supplemented medium is referred to as osteoblast differentiation medium or ObDM. Dexamethasone (DEX) has been previously demonstrated to induce osteoblast differentiation in vitro [7]. DEX (10–7 or 10–8 M final concentration) dissolved in ethanol or the same volume of ethanol (control) was added to ObDM to induce differentiation, and the media were changed two to three times per week until harvest.

Flow Cytometry and FACS Sorting of CD34+ Cells
CD34+ cells were harvested at indicated times using 1X trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA), rinsed, and incubated with appropriate antibodies. All cells (adherent and nonadherent) were collected and analyzed. Mouse IgG isotype controls were used to identify background or non-specific binding. Data shown represent net fraction (%) of CD34+ cells. To ensure that trypsin had no effect on the CD34 marker during the harvest, it was predetermined that trypsin-EDTA incubation for 5-10 minutes did not alter the ability of the CD34 antibody to label the cells (data not shown). The CD34+ cells were sorted using anti-CD38 antibody (Becton- Dickinson) to isolate CD34+/CD38– and CD34+/CD38+ cells. The sorted cells were plated in a limiting dilution manner, and scored as described below.

Limiting Dilution Analysis
Bone marrow (BM) low-density mononuclear (LDMN), CD34+ or CD34– cells were plated in flat-bottom 96-well plates at 50; 100; 500; 1,000; 2,500; and 5,000 cells/well. At least 18 wells for each starting cell number were plated per donor. ObGM was changed once per week, until adherent cells reached 70%-80% confluence, at which time ObDM + DEX (10–7 M) was added and changed twice per week until preosteoblasts were observed (between weeks 3 and 4). The wells were scored for the presence of preosteoblasts and further cultured until mineral deposits were observed (total culture time of five to six weeks) and stained with Alizarin red.

Osteoblast Histochemistry
For histochemical analyses, BM CD34+ cells were plated and cultured as described above, for "generation of osteoblasts from CD34+ cells: culture conditions." After 15 days, preosteoblast cells were subcultured and plated at a density of 4 x 103 cells per well in a 96-well plate in ObGM.

Alkaline Phosphatase   When the cells reached 70% confluence (two days later), they were treated with either ethanol (control) or 10–7 M DEX in ObDM for 4, 7, and 10 days before fixation. The cells were fixed with 2% paraformaldehyde in 0.1 M cacodylic acid, pH 7.4 at 4°C for 10 min, rinsed twice with 0.1 M cacodylic acid, and stored in the same buffer at 4°C until staining. The diazonium salt solution was prepared by dissolving one Fast violet B salt capsule in distilled water. Nathol AS-MX phosphate alkaline solution was added to the diluted diazonium salt solution to make an alkaline-dye mixture, and the mixture was added to each well of fixed osteoblasts and incubated at room temperature for 30 min, avoiding direct light. The wells were then rinsed with deionized water and photographed.

Osteonectin   When the cells reached 70% confluence, they were treated with either ethanol (control) in ObDM or DEX (10–7 M) in ObDM for 7, 10, and 14 days before fixation. The cells were fixed with 2% paraformaldehyde in 0.1 M cacodylic acid at 4°C for 10 min, rinsed twice with 0.1 M cacodylic acid, pH 7.4, and incubated with 10% normal goat serum for 15 min at room temperature. Either biotinylated anti-osteonectin monoclonal antibody (Biodesign International; Kennebunk, ME) or mouse IgG isotype control (10 ug/ml final concentration) was then added for two hours at room temperature. Streptavidin-ß-galactosidase and the X-Gal substrate were used for detection according to manufacturer's instructions (HistoMark Streptavidin-ß-Gal system, Kirkgaard and Perry Laboratories; Gaithersburg, MD).

Osteoblast Morphology: Transmission Electron Microscopy (TEM)
After the adherent cells had grown to 90% confluence, the cells were subcultured with trypsin/EDTA and replated into four-well chamber slides (LabTek, Nunc Inc.; Naperville, IL) at 7 x 104/ml. When osteoblast cells became 80%-90% confluent (five days later), ObDM containing 10–7 M DEX was added, and the medium was changed every three to four days for four weeks. When the cells had secreted mineral covering almost the entire surface of the chamber slide wells, the cells were fixed with 2.5% glutaraldehyde/1.6% paraformaldehyde in 0.1M cacodylic acid, pH 7.4. All processing was done in situ on the chamber slides. After 24 h of fixation, the cells were washed in 0.1M cacodylic acid, pH 7.4 overnight and post-fixed in 1% aqueous osmium tetroxide for one h at 4°. The cells were briefly rinsed, then dehydrated in ethanol. The specimens subsequently were infiltrated with an ethanol/epoxy resin mixture without the use of a transitional solvent. After a final addition of resin, the slides were polymerized and the blocks were trimmed and sectioned either in perpendicular or parallel orientation. The sections were stained with uranyl acetate and lead citrate prior to examination under TEM.

Osteoblast Functional Assays
For functional assays, BM CD34+ cells were plated, cultured, and subcultured as described above for "generation of osteoblasts from CD34+ cells: culture conditions" into 24-well plates.

Alkaline Phosphatase (AP) Activity   Three days after plating, when preosteoblasts were 70% confluent, ObDM containing either ethanol or DEX (10–7 M) was added for 4, 7, and 10 days. On those days, the cells were washed twice with 0.1M Tris, pH 8.0 and stored in 0.1M Tris/0.2% Triton at –80°C until assayed for AP activity. Cells from each well were scraped into 0.1M Tris/0.2% Triton-X 100, frozen in a dry ice-ethanol bath and thawed in a 37°C water bath for 5 minutes, two times. The AP activity in the lysates was measured using p-nitrophenyl phosphate disodium as substrate in 0.1M glycine, pH 10.5, and 1 mM MgCl2 with a p-nitrophenol standard. The protein content was measured by the Bradford method using Coomassie blue solutions and a BSA standard (Pierce; Rockford, IL). The enzyme activity was expressed as nanomoles of p-nitrophenol (per ml/mg protein). Each data point (mean ± SD) was obtained from triplicate wells.

PTH-Induced Cyclic Adenosine Monophosphate (cAMP) Increase   The cAMP enzyme-linked immunoassay (EIA) system was purchased from Amersham Life Science (Arlington Heights, IL), and human parathyroid hormone (PTH) (1-34) was purchased from Bachem Inc. (Torrance, CA). Preosteoblasts generated from BM CD34+ cells were subcultured using 1X trypsin-EDTA on day 19 into a 96-well plate (1 x 103 cells/well in ObGM). Five days after subcultivation, the cells were treated with ObDM containing either ethanol or DEX (10–7 M). Seven days after DEX treatment, the cells were rinsed twice with {alpha}-MEM containing 0.5% bovine serum albumin (BSA), and incubated at 37°C for five min with the same medium containing 0.5 mM isobutylmethylxanthine (incubation medium). Human PTH (1-34) (10–7 M) dissolved in the incubation medium was added, and the cells were further incubated at 37°C for five min. After washing twice with ice-cold phosphate buffered saline (PBS), 65% ice-cold ethanol was added to the well. The cells were scraped and transferred to a new microfuge tube. The remaining wells were rinsed with ice-cold 65% ethanol and pooled. The extracts were centrifuged at 2,000 g for 15 min at 4°C, and the supernatant from each tube was transferred to new tubes. The extracts were dried in a vacuum oven, and the level of cAMP was assayed using the EIA system, according to the manufacturer's instructions.

Alizarin Red Staining   Preosteoblast cells derived from BM and FL CD34+ cells were treated with either ethanol (control), or 10–7 M DEX in ObDM for 14 and 21 days before fixation. Cells were fixed with ice-cold 70% ethanol for one hour at 4°C. Cells were rinsed with distilled water and stained with Alizarin red (2%, pH 4.2) for 10 min at room temperature according to a published protocol [15]. After staining, cells were rinsed with PBS and photographed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of Preosteoblasts from BM CD34+ Cells in Culture
A schematic diagram of osteoblasts generated from BM CD34+ cells including the nomenclature used is illustrated in Figure 1. BM CD34+ cells ( Fig. 2A) cultured in ObGM generated many different cell types in vitro in the first seven days. The appearance of cell populations was as follows: Initially, many of the cells observed were blast-like ( Fig. 2B). After one week, the first medium change removed most of the nonadherent bone marrow cells. The remaining cells were adherent fibroblastic, polygonal, or macrophage-like in morphology ( Fig. 2C). With further culturing, osteoblast precursor and preosteoblast cells that were flat, elongated, and fibroblast-like started to take over the culture ( Figs. 2D and 2E). Subculturing of these preosteoblast cells following treatment with trypsin-EDTA further enriched the preosteoblast population, leaving behind the more tightly adherent macrophage population. By day 20, most of the cells resembled preosteoblasts, very flat in morphology and sheet-like in appearance ( Fig. 2F). These cultures containing preosteoblast cells were tested for the presence of adipocytes, chondrocytes, myocytes, and fibroblasts by Oil O red, alcian blue, myosin heavy chain antibody, and fibroblast-specific protein antibody staining, respectively. All of these stains were negative, demonstrating the absence of these cell types in the day 20 cultures (data not shown).



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  		Figure 1. A schematic diagram illustrating the culture method used to generate functional osteoblasts from BM CD34+ cells. The nomenclature used throughout the text to describe various intermediate populations is also represented. Abbreviations: BM = bone marrow; LDMN = low-density mononuclear cells; ObGM = osteoblast growth medium, described in Materials and Methods; ObDM = osteoblast differentiation medium.

 


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  		Figure 2. Appearance of human BM CD34+ cells in osteoblast-forming culture condition under a phase microscope. On day 0 (panel A), CD34+ cells are nonadherent and round in morphology. By day 3 (panel B), some elongated and irregular cells can be observed. After the first medium change on day 7 (panel C), adherent spindle- and polygonal-shaped cells can be seen. By day 10 (panel D), the adherent spindle-shaped cells are proliferating, along with some round cells that appear to be less adherent. By day 14 (panel E), a population of sheet-like cells (preosteoblasts) can be observed beneath some round and spindle-shaped cells. After subculture using trypsin-EDTA and re-establishment of culture (day 20), a relatively homogeneous population of flat, osteoblast-like cells can be seen (panel F). Panels A-E were photographed under the same magnification (size bar in panel E represents 50 microns) and panel F was photographed at a lower magnification (size bar in panel F represents 50 microns).

 
BM CD34+ Cells in Osteoblast Culture
To investigate the transition from CD34+ cells to preosteoblast cells, the expression of the CD34 antigen was followed over time by fluorescence-activated cell sorter analysis. Cultures were initiated with a population of cells that was 95% CD34+. By day 4 of culture, less than 10% of the cells were CD34+ ( Fig. 3). If this dramatic drop in CD34 expression were due to massive cell death, the total number of cells should be proportionally diminished ( Fig. 3). This was not the case. The total number of cells per well throughout the culture period did not change dramatically, indicating that cell death alone did not account for the presence of CD34– cells in culture. At each time point, the harvested cells were counted in a hemacytometer using trypan blue and in every case, >95% of the cells were viable. On the other hand, it is possible that some of the cells from the initial inoculum died and the remaining cells are converting to preosteoblasts, and the cell number coincidentally stays similar over time.



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  		Figure 3. CD34 expression by BM CD34+ cells in osteoblast culture (y-axis) and total cell number at the time of analysis (yy-axis) over time. BM CD34+ cells were cultured (inoculated with 250,000 cells/well). At each time point indicated, total cells (adherent and nonadherent) were harvested using trypsin-EDTA, counted using a hemacytometer and trypan blue (at all time points, <5% of cells were trypan blue positive), stained with CD34-PE, and analyzed by flow cytometry. Results shown are net CD34+ cells, using an isotype control antibody to subtract background binding. At all time points, cultures established from cells of four independent donors were analyzed.

 
Osteoblast Precursor Cell Frequency in BM LDMN, CD34+ and CD34– Cells
An osteoblast precursor cell is defined as a cell that can give rise to preosteoblasts ( Fig. 2F) that can secrete mineral upon further culturing with ObDM containing DEX. To determine the osteoblast precursor cell frequency, BM LDMN, CD34+, or CD34– cells were cultured in a limiting dilution assay (LDA) as described in Materials and Methods. The average frequencies of osteoblast precursor cells were 1/37,000 for LDMN, 1/5,000 for CD34+ and 1/33,000 for CD34– cells (Figs. 4A-4C). However, the total number of osteoblast precursor cells is higher (7.9-21 times, n = 4) in the CD34– population than in the CD34+ population ( Table 1). Preosteoblasts resulting from these LDAs were verified to be functional osteoblasts by further culturing in ObDM in the presence of DEX and Alizarin red staining of mineral (data not shown).



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  		Figure 4. Limiting dilution analyses of osteoblast precursor cells in human bone marrow LDMN (panel A), CD34+ (panel B), CD34– (panel C), and sorted CD34+/CD38+ (panel D) cells. Indicated cell populations were plated at initial plating densities (x-axis) cultured in ObGM and scored for osteoblast-like cells (between weeks 3-4) and graphed. The wells containing osteoblast-like cells were further cultured for two to three weeks in ObDM+DEX (10–7 M) to observe mineral formation. In all cases where osteoblast-like cells were observed, mineral secretion was also observed. For panels A-C, four independent donor cells were analyzed, and for panel D, two independent donor cells were analyzed.

 

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  		Table 1. Total number of osteoblast precursor cells present in CD34+ and CD34– populations of normal human bone marrow low-density mononuclear cells. Total cell number of CD34+ and CD34– populations were determined by using a hemacytometer and the osteoblast precursor cell frequencies of each population were determined by limiting dilution assay.
 
Osteoblast Precursor Cells are Found in the CD34+/CD38+ Fraction of the BM
To further fractionate the subpopulation of CD34+ cells that contain osteoblast precursor cells and to reduce the possibility that the small proportion of non-CD34+ cells in the CD34+ cell population was giving rise to osteoblasts, the CD34-selected cells were re-stained with various antibodies and sorted. Of the sorted populations, including CD34+/cw90+, CD34+/cw90–, CD34+/CD38–, and CD34+/CD38+ cells, the only population which consistently gave rise to osteoblasts was the CD34+/CD38+ population (n = 3 for each marker). The osteoblast precursor cell frequency in the CD34+/CD38+ population was 1/6,700 ( Fig. 4D), similar to that in the CD34+ population (1/5,000). Since most of the cells in the CD34+ population were CD38+, these results indicate that the osteoblast precursor cells remain in the CD34+/CD38+ population.

Preosteoblasts Generated from BM CD34+ Cells Increase Alkaline Phosphatase and Osteonectin Expression upon Differentiation
Skeletal-specific alkaline phosphatase and osteonectin are well-known markers of osteoblast differentiation [6, 7, 10, 11, 16]. Treatment of preosteoblasts generated from BM CD34+ cells with DEX increased the expression of alkaline phosphatase and osteonectin over time ( Figs. 5 and 6). Although the control cultures expressed baseline levels of alkaline phosphatase and osteonectin, levels of these proteins are clearly and significantly elevated in these cultures treated with DEX.



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  		Figure 5. Alkaline phosphatase histochemical stain of preosteoblast cells before DEX treatment (panel A), treated with either ethanol (panels B-D) or 10–7 M DEX (panels E-G) in ObDM for 4 (panels B and E), 7 (panels C and F), and 10 (panels D and G) days are shown (size bar = 50 µm).

 


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  		Figure 6. Osteonectin immunohistochemical stain of preosteoblast cells before DEX treatment (panel A), treated with either ethanol (panels B-D) or 10–7 M DEX (panels E-G) in ObDM for 7 (panels B and E), 10 (panels C and F), and 14 (panels D and G) days is shown (size bar = 50 µm).

 
Ultrastructure of Osteoblasts Generated from BM CD34+ Cells
To investigate the ultrastructural morphology of osteoblasts generated from CD34+ cells, TEM was performed. The overall cell size and shape (cuboidal) were consistent with osteoblasts observed in vivo [17]. The cell displayed in Figure 7A has an acentric nucleus placed toward one side of the cell and shows uneven cell surface. A well-defined Golgi apparatus adjacent to the nucleus is present on one side. Lack of prominent and extensive rough endoplasmic reticulum is probably due to the lengthy culture time necessary to accumulate sufficient mineral. However, there are numerous free ribosomes, vacuoles, and dense-staining lysosomes in the cytoplasm of the cell shown, features consistent with an osteoblast [18]. Figure 7B illustrates the mineral accumulation among collagen fibrils [18] found near the cell shown in Figure 7A.



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  		Figure 7. Transmission electron micrograph of osteoblasts generated from BM CD34+ cells (panel A) and of mineral deposition along collagen fibrils (panel B). Size bars in panels A and B represent five and one µm, respectively.

 
Preosteoblasts Generated from BM CD34+ Cells Increase Alkaline Phosphatase Activity and PTH-Induced cAMP Production upon DEX Treatment
The increase in skeletal specific alkaline phosphatase activity [6, 7] and the increase in PTH-induced cAMP production [7] accompanying osteoblast differentiation have been well described. Osteoblasts generated from CD34+ cells treated with DEX show an increase in skeletal specific alkaline phosphatase activity with increased time of DEX exposure ( Fig. 8A). This increase in alkaline phosphatase activity was observed in cells from all donors tested (n = 3), and cells from the donor that represented the median response are shown on Figure 8A. The increase in cAMP production in response to PTH stimulation of differentiated and control osteoblasts is shown in Figure 8B. Preosteoblasts stimulated to differentiate into osteoblasts with 10–7 M DEX demonstrated a significant increase in cAMP production in response to 10–7 M PTH. This increase was not seen in control cells, as expected.



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  		Figure 8. (A) Alkaline phosphatase activity of BM CD34+ cell-generated preosteoblast cells increases upon DEX treatment. Preosteoblast cells generated from BM CD34+ cells were treated with either ethanol (control) or DEX (10–7 M) for 4, 7, and 10 days. Total enzymatic activity was normalized to total protein concentration for each time point analyzed. For each data point, triplicate wells were analyzed. The data represented are obtained from cells of one donor and similar results were obtained with other donor cells (n = 3). (B) PTH-induced cAMP level of BM CD34+ cell-generated preosteoblast cells increases upon DEX treatment. Preosteoblast cells generated from BM CD34+ cells were treated with either ethanol (control) or DEX (10–7 M) for seven days, stimulated with PTH (10–7 M) for five min and analyzed for cAMP levels as described in Materials and Methods.

 
Osteoblasts Generated from BM CD34+ Cells Increase Mineral Secretion upon Differentiation
Mineral secretion is a terminal and definitive differentiation function specific to osteoblasts [6, 7, 11]. Osteoblasts from BM and fetal liver (FL) CD34+ cells secrete hydroxyapatite into the extracellular matrix in response to DEX addition ( Fig. 9). Alizarin red specifically stained the mineral in cultures treated with DEX but not in control cultures. Interestingly, CD34+ cells from bone marrow and fetal liver, but not from cord blood (n = 5) or peripheral blood (n = 7), gave rise to mineral-secreting osteoblasts under these culture conditions (data not shown).



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  		Figure 9. Alizarin red staining of mineral secreted by osteoblasts generated from BM or fetal liver (FL) CD34+ cells upon DEX treatment. Osteoblast-like cells generated from BM CD34+ cells (panels D-F) and FL CD34+ cells (panels A-C) were treated with either ethanol (panels A and D) or DEX (10–7 M) for 14 (panels B and E) and 21 (panels A, C, D, and F) days. On the indicated days, the cultures were fixed, stored at 4°C, and when all time points were collected, stained at the same time. The size bar in panel F represents 50 µm for all panels.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Bone is a dynamic organ that is under constant remodeling, and osteoblasts play a major role in this remodeling process. To date, information on human osteoblast progenitor cells of the bone marrow is relatively limited. In the last several years, it has become increasingly clear that hematopoiesis and the stromal environment in which osteoblast progenitor cells are thought to be found are closely related. Reports in the literature indicate that there may also be some overlap between the hematopoietic and stromal cells, including cytokine and cytokine receptor expression by early precursor cells. These overlaps may not be surprising considering the close proximity between hematopoiesis and the stromal environment.

Our results show that CD34+ cells from normal human bone marrow aspirates contain osteoblast precursor cells that can be cultured to become morphologically and functionally normal osteoblasts. This finding confirms other reports describing the presence of CFU-F in CD34+ cells [13] and the presence of osteoblast precursor cells in STRO-1+ cells [16], but contradicts another study in which osteoblasts could not be generated from BM CD34+ cells [10]. The difference between that study and ours may be due to different culture conditions, such as serum-free versus serum-containing, different media components and length of culture time. We have used the most osteogenic permissive system available [6, 7, 11], containing 10% FBS (preselected) to determine which bone marrow subpopulations contain osteoblast precursor cells. Whether a selected lot of FBS contains novel osteogenic factors that are absent in unselected lots of FBS is presently unknown.

Although the frequency of osteoblast precursor cells was increased in the CD34+ population compared to the CD34– and LDMN populations, the total number of osteoblast precursor cells is still relatively low in the CD34+ population due to the low number of CD34+ cells present in BM LDMN cells. The significance of CD34 expression in cells capable of giving rise to osteoblasts is not clear at this time.

STRO-1 is an antibody that was generated by injection of CD34+ cells into mice and which has been studied in detail to identify stromal precursor cells [19]. It has been reported that about 5% of BM cells are CD34+/STRO-1+ [13]. In our studies, less than 0.5% (average 0.403 ± 0.104%, n = 4) of BM CD34-selected cells are also STRO-1+ (data not shown). The difference in these numbers may be due to the cell population analyzed, one being BM mononuclear cells and the other being CD34-selected cells. Due to the low percentage of double-positive CD34+/STRO-1+ cells, it was not possible to isolate this population to conduct further studies. However, the fact that STRO-1 antibody was generated by injecting CD34+ cells also implies that CD34+ cells contain stromal precursor cells.

It has been proposed that the frequency of osteoblast precursor cells decreases with increased donor age. In this study, bone marrow aspirates were collected from relatively young healthy adult donors (23.4 ± 3.6 years, n = 12). Most studies that correlate osteoblast precursor cell frequency with age were conducted using animals [20, 21]. One study using human marrow cells in diffusion chamber cultures demonstrated a possible decline in osteoblast precursor cells with donor age, but the possibility that the result was due to a decline in the capacity of osteoblasts to undergo terminal differentiation could not be ruled out [22]. Therefore, whether the osteoblast precursor cell frequency is altered with age still remains to be demonstrated.

The mechanisms and signals that specifically regulate osteoblast commitment, proliferation, and differentiation are still unclear. Recently, a report by Scutt and Bertram demonstrated that PGE2 induced osteoblast precursor cells to transition from nonadherent to adherent cells [23], implicating an important role for PGE2 in early osteoblast progenitor cell commitment. Other investigators have reported enrichment of osteoblast progenitor cells by positive [10] or negative [11] selection from human bone marrow. In addition, it has been reported that 5-fluorouracil treatment of the mouse bone marrow increases osteoblast precursor cell frequency 12-fold [18]. It is possible that the osteoblast lineage is similar to the hematopoietic lineages, where myeloablation results in upregulation of endogenous osteoblast-specific factors. As these human in vitro osteoblast systems become increasingly refined, they may be used to elucidate the early events of osteogenesis and also to screen for possible novel osteoblast-specific factors that may have therapeutic potential for bone-related diseases, such as osteoporosis.


   Acknowledgments
 
The authors would like to thank Francois Périer, Steve Coats, Janet Nichol, and Colin Dunstan for critical readings of the manuscript, Jamie Wang for technical contributions and support, Kim Warren of Poietic Technologies, Inc. for providing donor age and % CD34+ cells, Martha Hokom and Xiao Ling Xiong for providing bone marrow CD34– cell-derived stromal cultures, and Dr. George Morstyn for support of this project.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication July 15, 1997.




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