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First published online June 21, 2007
Stem Cells Vol. 25 No. 10 October 2007, pp. 2575 -2582
doi:10.1634/stemcells.2007-0204; www.StemCells.com
© 2007 AlphaMed Press

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TECHNOLOGY DEVELOPMENT

Clinical-Scale Expansion of a Mixed Population of Bone Marrow-Derived Stem and Progenitor Cells for Potential Use in Bone Tissue Regeneration

James E. Dennisa, Kelly Esterlyb, Amad Awadallaha, Christopher R. Parrishb, Gregory M. Poynterb, Kristin L. Goltryb

aDepartment of Orthopaedics, Case Western Reserve University, Cleveland, Ohio, USA;
bAastrom Biosciences Inc., Ann Arbor, Michigan, USA

Key Words. Bone marrow • Osteogenesis • Bioreactor • Ex vivo expansion

Correspondence: James E. Dennis, Ph.D., Department of Orthopaedics, University Hospitals of Cleveland, 6th Floor Hanna Building, 11100 Cedar Avenue, Cleveland, Ohio 44106, USA. Telephone: (216) 368-3567; Fax: (216) 368-1332; e-mail: James.Dennis{at}case.edu

Received March 26, 2007; accepted for publication May 25, 2007.
First published online in STEM CELLS EXPRESS   June 21, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Preclinical and clinical studies have demonstrated the ability of bone marrow derived stem and progenitor cells to regenerate many tissues, including bone. Methods to expand or enrich progenitors from bone marrow are common; however, these methods include many steps not amenable to clinical use. A closed automated cell production culture system was developed for clinical-scale ex vivo production of bone marrow-derived stem and progenitor cells for hematopoietic reconstitution. The current study tested the ability of this bioreactor system to produce progenitor cells, termed tissue repair cells (TRC), possessing osteogenic potential. Three TRC formulations were evaluated: (a) cells cultured without exogenous cytokines (TRC); (b) cells cultured with exogenous cytokines (TRC-C); and (c) an adherent subset of TRC-C (TRC-CAd). Starting human bone marrow mononuclear cells (BM MNC) and TRC products were characterized for the expression of cell surface markers, in vitro colony forming ability, and in vivo osteogenic potential. Results showed significant expansion of mesenchymal progenitors (CD90+, CD105+, and CD166+) in each TRC formulation. In vivo bone formation, measured by histology, was highest in the TRC group, followed by TRC-CAd and TRC-C. The TRC product outperformed starting BM MNC and had equivalent bone forming potential to purified MSCs at the same cell dose. Post hoc analysis revealed that the presence of CD90+, CD105+, and CD166+ correlated strongly with in vivo bone formation scores (r2 > .95). These results demonstrate that this bioreactor system can be used to generate, in a single step, a population of progenitor cells with potent osteogenic potential.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Efficient tissue repair or regeneration remains a significant challenge. In the orthopedic field, the use of autologous bone grafting remains the clinical practice standard of care, although it is limited in the availability of graft material and donor site morbidity [1]. Another approach to the treatment of nonunion fractures is to harness and expand the inherent osteogenic potential of bone marrow cells. Bone marrow was identified as a source of osteoprogenitor cells as early as 1867 [2], and the first heterotopic transplantation showing bone formation in an animal model was described in 1869 [3] and quickly confirmed in the following year [4]. Bone marrow has since been shown to contain cells with multiple mesenchymal lineages [5–13] and, under specific culture conditions, a subset of marrow cells has been shown to express additional phenotypic markers from the ectodermal and endodermal lineages [14]. Recent results have shown that whole bone marrow combined with ceramic carriers can promote bone repair in animal models [15, 16]. A more recent development shows the use of the carrier matrix to first concentrate the marrow-derived osteoprogenitor cells while it also functions as a scaffold for bone repair [17]. However, none of these methods using whole bone marrow seek to expand the osteoprogenitor cell pool and are therefore limited to the number of osteogenic donor cells that can be harvested from the patient. Patient-specific cell therapy, in many cases, requires large numbers of cells to replace damaged or diseased tissue. For large bone defects or nonunion fractures, this number may be insufficient for the complete and timely repair and recovery of skeletal function. Therefore, for many cases, methods may be required to increase the population of osteogenic cells through ex vivo expansion techniques. Culture-expanded marrow-derived mesenchymal stem and progenitor cells have been shown to repair segmental bone defects in animal models [18, 19], including human MSCs implanted into athymic rats [20]. The expansion method for mesenchymal stem cells typically involves multiple steps in an open process and can take 2–3 weeks. For clinical applications, a simple, safe, reproducible means of effectively expanding the osteogenic pool of cells within the bone marrow is needed.

We have previously found that single-pass perfusion (SPP) technology results in significant expansion of primary human cells [21–25], particularly increases in stem and progenitor pools, possessing enhanced functional abilities. In SPP, culture medium is continuously replaced by fresh medium at a slow, controlled rate without disturbance or removal of cells, enabling optimal exchange of nutrients and metabolic by-products and maintenance of the microenvironment. Specifically, the importance of medium perfusion has been demonstrated not only on the improved productivity and longevity of BM cultures [21] but also on the metabolic activity and growth factor production rates of marrow stromal cells [23].

Application of SPP technology led to the development of an automated perfused bioreactor system for the clinical-scale expansion of human primary cells [26]. In this bioreactor system, oxygenation of the culture is decoupled from medium perfusion, allowing optimization of the medium exchange rates for optimal culture performance. Bone marrow mononuclear cells (BM MNC) cultured in the bioreactor go through a major change in the composition of cell populations, resulting in a mixed lineage cell product, termed tissue repair cells (TRCs), that has been used to generate patient-specific cell therapy for hematopoietic reconstitution [27, 28]. Here, the potential of TRCs is being investigated for its clinical application to bone repair.

TRCs are a mixed cell population containing cell phenotypes normally present in the starting BM MNC, including cells of hematopoietic, mesenchymal, and endothelial lineages, but at different frequencies. Several modifications of the cell expansion and cell harvesting protocols were investigated to identify culture conditions that would more efficiently enrich for osteogenic subpopulation of cells. In this study, both in vitro and in vivo bone formation results demonstrate that this system is an effective means for expanding the osteoprogenitor cell populations in marrow, and that several modifications of culture harvesting techniques or culture components have resulted in enhanced in vivo bone formation found to be equivalent to more purified MSC populations. In addition, the frequency of several cell surface markers correlates with in vivo bone scores.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Bone Marrow Mononuclear Cells
Fresh BM MNC from normal donors were purchased from Poietics Inc. (Gaithersburg, Maryland, http://www.lonzabioscience.com) and assessed by flow cytometry, in vitro colony assays, and in vitro differentiation assays as described below. The donors ranged in age from 18–45 years old (average 25.6); 62% were male and 38% were female, with 75% African-American, 21% Caucasian, 3% Asian, and 1% Hispanic. These BM MNC were then used to initiate cultures to generate TRCs. Human bone marrow aspirates were obtained from University Hospitals of Cleveland with Institutional Review Board approval and within NIH guidelines for research involving human subjects. BM MNC were isolated from the bone marrow aspirate using a Percoll gradient and then used to initiate MSC cultures. In some cases, BM MNC were cryopreserved at approximately 3.3 x 107 cells per milliliter in cryogenic vials in a final concentration of 7.5% dimethyl sulfoxide and 20% fetal bovine serum (FBS) in Iscove's modified Dulbecco's medium (IMDM). The vials were placed in an insulated container (Nalgene, Rochester, NY, http://www.nalgenunc.com) overnight at –80°C and transferred to liquid nitrogen. Vials containing either fresh BM MNC or in some cases frozen BM MNC shipped overnight.

TRC Cell Production
After assessing cell concentration using the Coulter Z2 (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com), BM MNC were inoculated at 2–3 x 108 cells per cell cassette (includes bioreactor and medium reservoir) [26]. Prior to inoculation, bioreactors were primed with long-term bone marrow culture (LTBMC) medium consisting of IMDM, 10% fetal bovine serum, 10% horse serum, 5 µM hydrocortisone, gentamicin sulfate, and vancomycin. The bioreactor is designed such that medium flow rate can be adjusted at any time during the culture process within an operating range of 10%–500% volume medium exchange per day depending on the needs of the culture. The entire volume of the culture area is approximately 280 ml. For TRC production, LTBMC was passed through the bioreactor at a controlled ramped perfusion schedule starting at 0% volume medium exchange from day 1 to day 3, 25% exchange per day from days 3 to 8, and 75% exchange per day from day 8 until harvest on day 12. The cultures were maintained at 37°C with 5% CO2 and 20% O2 in the incubator for the duration of the culture. After harvest by trypsinization (0.025% trypsin-EDTA in 0.9% sodium chloride), TRCs were shipped overnight at room temperature in LTBMC for analysis of in vivo bone formation at Case Western Reserve University. An aliquot of TRCs was also analyzed by flow cytometry and in vitro assays as described below.

Cell Production of TRC Cultured with Exogenous Cytokines
BM MNC were cultured as described above for TRCs except that exogenous growth factors, 25 ng/ml flt3 ligand (Immunex, Seattle, http://immunex.com), 0.1 U/ml erythropoietin (Amgen, Thousand Oaks, CA, http://www.amgen.com), and 5 ng/ml PIXY321 (Immunex) were present in the culture medium. Again, cells were harvested by trypsinization and analyzed as described.

Cell Production of an Adherent Subset of TRC Cultured with Exogenous Cytokines
BM MNC were cultured as described above for TRC cultured with exogenous cytokines (TRC-C). To obtain the plastic adherent subpopulation from TRC-C, the nonadherent cells were removed from the bioreactor by draining and then discarded. The remaining adherent cells in the bioreactor were then trypsinized and analyzed as described. This adherent cell population includes both plastic adherent stromal cells and hematopoietic cells closely associated with the stroma.

MSC Culture
BM MNC were placed in 10% FBS in Dulbecco's modified Eagle's medium (DMEM) and plated at 1.0 x 107 cells per milliliter in 100-mm tissue culture dishes. Medium was exchanged twice weekly, and cells were harvested by incubation in 4 ml of trypsin/EDTA for 5 minutes at 37° C followed by the addition of 2 ml of normal calf serum, centrifugation, and resuspension in serum-free DMEM.

Flow Cytometry
Starting BM MNC and the TRC expansion products were washed and resuspended in 1x Dulbecco's phosphate buffered saline (PBS; Gibco, Grand Island, NY, http://www.invitrogen.com) containing 1% bovine serum albumin. Tubes containing 106 cells in 0.5 ml were stained on ice with various combinations of fluorescently conjugated monoclonal antibodies. Viability was determined by 7-aminoactinomycin D (7AAD) (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com); 7AAD only enters membrane-compromised cells and binds to DNA. Several sets of markers commonly used to identify mesenchymal cells were used, including CD90+/CD14–, CD105+/CD166+/CD14–/CD45–, and CD146+. Cells were stained with PC5-conjugated anti-CD90 (Thy1) antibodies and fluorescein isothiocyanate (FITC)-conjugated anti-CD14 (Beckman Coulter), CD105-FITC (Serotec Ltd., Oxford, U.K., http://www.serotec.com), CD166-phycoerythrin (PE) (BD Biosciences, San Diego, http://www.bdbiosciences.com), FITC-conjugated anti-CD14 and anti-CD45 (Beckman Coulter), or CD146-PE (Beckman Coulter). Hematopoietic progenitor cells were assessed by staining cells with either CD34-PE (Beckman Coulter) or IgG-PE (control; Beckman Coulter) monoclonal antibody along with a cocktail of lineage (lin)-specific FITC-conjugated antibodies: CD3, CD11b, CD15, CD20, and glycophorin-A (Gly-A [CD235a]) (Beckman Coulter). Myeloid progenitors were stained with CD13-FITC (Beckman Coulter) along with a lin B cocktail of PE-conjugated antibodies: CD3, CD11b, CD15, CD20, and Gly-A (CD235a) (Beckman Coulter). Erythroid cells were assessed by staining with FITC-anti-Gly-A (CD235a). After 15 minutes, cells were washed and resuspended in 0.5 ml of PBS/bovine serum albumin (BSA) for analysis on the Epics XL-MCL (Beckman Coulter) flow cytometer. Results are presented as percent of cells positive for specific markers after subtracting the value of the isotype controls. All isotype control levels were less than 0.5%. Cells were sorted for specific phenotypes using the Epics Altra (Beckman Coulter).

Colony Forming Unit-Fibroblast Assay
Cells were plated in 1 ml of LTBMC (see above) in 35-mm tissue culture treated dishes. For BM MNC, 150,000 and 500,000 cells were plated per dish. For TRCs and MSCs, 100 and 400 cells were plated per dish. Cultures were maintained for 8 days at 37°C in a fully humidified atmosphere of 5% CO2 in air. Colony forming unit-fibroblast (CFU-F) colonies were then stained with Wright-Giemsa, and colonies with greater than 20 cells were counted as CFU-F.

Colony Forming Unit-Granulocyte/Macrophage Assay
Cells were inoculated in colony assay medium containing 0.9% methylcellulose (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 30% FBS, 1% BSA, 100 µM 2-mercaptoethanol (Sigma), 2 mM L-glutamine (Gibco), 5 ng/ml PIXY321, 5 ng/ml granulocyte colony-stimulating factor (Amgen), and 10 U/ml erythropoietin. BM MNC were plated at 10,000 and 20,000 cells per milliliter, and TRCs were plated at 1,500 and 3,000 cells per milliliter. Cultures were maintained for 14 days, and colonies with greater than 50 cells were scored as colony forming unit-granulocyte/macrophage (CFU-GM).

Osteogenic Differentiation Assays
Cells were cultured for 1–2 weeks in 35-mm dishes containing either control osteogenic (OS–) medium (DMEM with 10% FBS) or OS+ medium (DMEM containing 10% FBS, 100 nM dexamethasone, 10 mM β-glycerophosphate, and 0.05 mM L-ascorbate-2-phosphate) at a concentration of 10,000–20,000 cells per cm2. Osteogenic differentiation was assessed by cell morphology, expression of alkaline phosphatase (AP), and formation of a mineralized matrix by calcium deposition. AP activity present in the differentiated culture was quantified by measuring the rate of conversion of p-nitrophenyl phosphate to p-nitrophenol (Sigma) and measuring absorbance at 410 nm. Enzyme activity is expressed as nmoles of p-nitrophenol/minute and p-nitrophenol/minute per 106 plated cells. Calcium was quantified following the procedure provided in the Calcium Quantitative Kit (Pointe Scientific Inc., Canton, MI, http://pointescientific.com). Briefly, osteogenic cultures were lysed with 0.5 N HCl, and lysates were collected into microcentrifuge tubes. After vortexing, each sample was shaken at 500 rpm for 4 hours at 4°C. After centrifugation at 1,000g in a microcentrifuge, supernatants were collected and assayed for the presence of calcium by measuring absorbance at 570 nm.

In Vivo Bone Formation
TRCs, MSCs, or cell-free DMEM were incubated with fibronectin-coated porous calcium phosphate ceramic blocks (Zimmer, Warsaw, IN, http://www.zimmer.com), approximately 27 mm3 in volume, and then implanted subcutaneously into the backs of CB-17 severe combined immunodeficient (SCID) mice (CB-17/IcrCrl-SCID-BR; Charles River Laboratories, Wilmington, MA, http://www.criver.com). Cell loading concentrations in these studies were 5, 10, 25, and 50 million cells per milliliter. For each experiment, mice were implanted with six sets of ceramics, specifically ceramics loaded with TRCs at cell concentrations of 5, 10, 25, and 50 million cells per milliliter, ceramics loaded with MSCs at 5 million cells per milliliter, and ceramics incubated in medium alone. The location of the implanted ceramics was systematically changed to avoid any potential location-specific differences in bone formation. Ceramic implants were harvested from host SCID mice after 6 weeks, fixed in formalin, decalcified, embedded in paraffin, and serially sectioned. Sections were stained with Mallory's Heidenhain, and visually blinded specimens were scored for presence of bone within pores. The method of estimating bone formation does not estimate bone volume, rather, the bone score is defined by the number of ceramic pores that contain some bone out of the total number of pores containing cells on a scale from 0–5, where 0 = no bone detected, 1 = >0%–20% of pores, 2 = >20%–40%, 3 = >40%–60%, 4 = >60%–80%, and 5 = >80% of pores have bone. This is a subjective bone scoring method that is a modification of a method used in this laboratory that has been shown to correlate strongly (r2 = 0.77) with direct measurements of bone area percent [29]. Groups were compared statistically by analysis of variance and Mann-Whitney post hoc test for significance among groups.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Phenotypic Characterization of TRC Formulations
Each preparation of BM MNC was assessed for the expression of cell surface markers and for the ability to form clonogenic colonies. The TRC-C formulation, cultured in the presence of cytokines, was previously shown to reconstitute the hematopoietic system in patients and contains both hematopoietic and stromal elements [27, 28]. Variations of this product, including the TRC-C adherent layer alone (TRC-CAd) or TRCs generated without exogenous factors (TRC), were also tested. The frequencies of selected cell types under different culture and harvesting conditions are shown in Table 1.


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  		Table 1. Frequency (%) of cell phenotypes in BM MNC and different TRC products

 
The greatest frequency of CFU-GM, Gly-A+, and CD13+ hematopoietic cells is in the TRC-C preparations, followed by the TRC-CAd, and lastly by the TRC preparation. The most dramatic difference is observed in the TRC cell product, where the frequency of mesenchymal cells, including CFU-F, CD90+, CD146+, and CD105+/166+ populations, is dramatically higher than in the other culture conditions. TRCs had more than four times the frequency of CD90+ cells compared with the next highest group (TRC-CAd) and approximately five times the frequency of CD105+/166+ cells compared with TRC-CAd. Cell sorting experiments showed that all CFU-F were found in the CD90+ fraction (3.2 ± 0.8 per 100 nucleated cells), whereas none were found in the CD90– fraction.

The fold expansion of different subpopulations was assessed by dividing the total number of output cells by the total number of input cells for each bioreactor. The mean and standard deviation for each are shown in Table 2. The TRC-C culture conditions result in the greatest expansion of total nucleated cells and hematopoietic cells such as CFU-GM, CD13+, Gly-A+, CD34+, and CD14+ cells, whereas TRC culture conditions promote the expansion of stromal components (CD90+, CD105+, CD166+, CD146+, and CFU-F) and, at the same time, a decreased expansion of the hematopoietic elements and total nucleated cells. The high degree of variation seen in the fold expansion is primarily a result of high variability of the donor input frequencies.


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  		Table 2. Fold expansion of cell types

 
In Vitro Osteogenic Potential of TRC Formulations
Each preparation of BM MNC was assessed for its ability to differentiate along the osteogenic lineage. TRCs were incubated in OS– (control) or OS+ medium for 12 days and assessed for calcium deposition and alkaline phosphatase activity. Since osteogenic potential varies from donor to donor, the results from one representative experiment comparing TRC formulations generated from the same bone marrow donor are presented in Table 3. TRCs had the highest levels of osteogenic differentiation, followed by TRC-CAd and TRC-C. This relative trend has been observed in other experiments comparing either TRCs and TRC-C or TRCs and TRC-CAd. Results from cell sorting experiments showed over 500 times more calcium accumulation in the CD90+ fraction (7.30 µg per dish) compared with the CD90– fraction (0.01 µg per dish) in in vitro osteogenic assays.


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  		Table 3. In vitro osteogenic potential of TRC

 
In Vivo Osteogenic Potential of TRC Formulations
An in vivo assay for bone formation was used to assess the osteogenic potential of TRCs. In one set of three experiments, TRC-C (cell population cultured with cytokines) was compared with TRC-C Ad to determine whether osteogenic potential is enriched in the adherent layer. TRC-C contained both nonadherent and adherent cells, while TRC-CAd is the adherent population of TRC-C. For each experiment, a single source of BM MNC was used to inoculate two different bioreactors that were harvested with or without nonadherent cells. The samples were implanted, harvested after 6 weeks, prepared for histologic examination, and scored for bone formation as described in Materials and Methods. An example of the histologic results is shown in Figure 1, where 1A and 1B show results from the TRC-CAd group and 1C and 1D from the TRC-C group, both loaded at 5.0 x 107 cells per milliliter. Many of the pores here are positive for bone formation and are comparable to the results for MSC, shown in Figure 1E and 1F. Most empty control ceramics were completely negative (Fig. 1G, 1H). The individual results of the bone scores from all three experiments showed the same general trend of osteogenic response (bone score) after 6 weeks in vivo. The results depicted in Figure 1 are shown as examples of how the in vivo histologic data look and are not intended to be representative of the quantitative results. For quantitative analysis, the bone score data from all of the samples were combined and are shown in tabular form in Table 4. The TRC-C and TRC-CAd at each cell loading concentration were compared with each other using the Mann-Whitney U test. The results showed significantly higher bone scores for the TRC-CAd over the TRC-C at a cell loading concentration of 5.0 x 107 cells per milliliter (p = .034), and the same trend of the TRC-CAd showing a higher bone score than identically loaded TRC-C samples was observed for the other three dilutions. The medium controls showed only sporadically positive bone scores, whereas the MSC scores were generally high. In two of the experiments, not all of the implanted animals survived the entire 6 weeks, thus resulting in differences in total numbers for the TRC-C and TRC-CAd groups and some differences in the numbers of ceramics placed at different locations in the host animal. In total, 3 animals were lost out of 36 that were implanted with ceramics.


Figure 1
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  		Figure 1. Histologic sections of ceramics harvested 6 weeks postimplantation. Ceramics loaded with an adherent subset of tissue repair cells cultured with exogenous cytokines (A, B) and nonadherent tissue repair cells cultured with exogenous cytokines (C, D) at 1.0 x 107 cells per milliliter. The darkly stained regions (arrows) are bone, which is found adjacent to the ceramic pore edge. Positive control MSCs are shown in (E, F), and empty controls are shown in (G, H). Abbreviations: DCR, demineralized ceramic residue; P, pore.

 


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  		Table 4. In vivo bone scores: TRC-C vs. TRC-CAd

 
In a second set of experiments, TRCs, cultured without exogenous cytokines and highly enriched for mesenchymal cells, were compared with TRC-Cs and enriched for both hematopoietic and mesenchymal cells. For each run, a single source of BM MNC was used to inoculate two different bioreactors that were cultured either in the presence of growth factors (TRC-C) or the absence of growth factors (TRC). Again, the samples were implanted, harvested after 6 weeks, prepared for histologic examination, and scored for bone formation as described in Materials and Methods. Results in Figure 2 show that, for each donor, the ceramics implanted with TRCs scored highest for bone formation. Bone scores were equivalent between TRCs and MSCs at the same cell-loading dose. A dose response was not observed for TRC, as the lowest loading cell dose of 5 x 106 cells per milliliter gave maximal bone score. In both bone marrow samples, there was a low level of positive scores in the empty controls. Unexpanded BM MNC and all dilutions of TRC-C had bone scores indistinguishable from those of the negative controls. These scores were lower than the scores in TRC-C samples from the previous three experiments (Table 3) and are attributed to variability in donor bone marrow.


Figure 2
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  		Figure 2. Bone scores for two separate experiments in ceramics loaded with tissue repair cells cultured with exogenous cytokines and TRCs. Ceramics were harvested at 6 weeks postimplantation and scored for bone on a 0–5 scale. The TRC samples showed high bone scores even at the lowest cell loading concentration, whereas TRC-C samples from these two donors showed very low scores. Abbreviations: BM MNC, bone marrow mononuclear cells; TRC, tissue repair cell cultured without exogenous cytokines; TRC-C, tissue repair cells cultured with exogenous cytokines.

 
Correlation of Mesenchymal Cells with In Vivo Bone Formation
Because TRCs showed the highest levels of differentiation in vitro and bone formation in vivo, it was hypothesized that the frequency of cells enriched in TRC preparations might correlate with in vivo bone scores. To test for this, the in vivo bone scores were plotted against the frequency of each of the cell types (Fig. 3). Not surprisingly, all of the cells within the stromal or mesenchymal category (CD105+/CD166+, CD90+, and CFU-F) correlated highly with in vivo bone formation. Even though the degree of CD105+ cell expansion was not dramatically different between TRC and TRC-C preparations (less than twofold difference; Table 2), there was also a clear correlation of CD105+ cell numbers with in vivo bone scores. This suggests that the overall frequency of specific cell types, rather than their fold expansion, seems to be a more important parameter in predicting the osteogenic potential of the TRC cell product.


Figure 3
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  		Figure 3. Correlation graph between cumulative bone score (summed mean values from ceramics loaded at four different densities) and frequency of CFU-F and selected cell surface markers determined by flow cytometry. Regression curves and r2 values were derived using a polynomial third order equation and goodness of fit. Abbreviation: CFU-F, colony forming unit-fibroblast.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Overall, these results show that BM MNC can be cultured in an automated closed-system bioreactor, and the resulting cell products possess a diverse range of cell phenotypes with the potential to differentiate down the osteogenic lineage under the right conditions. The methods of expansion and harvesting of BM MNC-derived cell products lead to unique cell products with differing potential for in vivo bone formation. In addition, examination of several cell surface markers revealed a strong correlation between the frequency of cell surface markers CD105+, CD166+, and CD90+ and in vivo bone formation scores when implanted with a ceramic matrix material.

Ex Vivo Expansion
The bioreactor system was initially developed for the expansion of hematopoietic stem cells within the context of the marrow supportive stromal cell microenvironment. The expansion was a single-step 12-day process within a closed system, and cells were exposed to a specific medium perfusion schedule established to enhance stem and progenitor cell output. This process did not include removal of nonadherent cells early in the culture process as is typical for more purified MSC preparations. In each formulation, the same cell phenotypes were present as the starting BM MNC population, although the frequencies of these cells were different depending on culture conditions.

The cell culture medium used to generate TRCs contains both horse and fetal bovine serum. Prior to administering a cellular therapy to patients, components such as animal sera as well as proteolytic reagent compounds must be removed or minimized. Therefore, washing the cell product extensively after harvest is required to reduce or eliminate serum proteins and trypsin from the cell product. A particular risk with the use of animal derived products is exposure of the cells to infectious agents. Since 1993, the Food and Drug Administration (FDA) has recommended that bovine-derived material from cattle that have resided in or originated from countries where bovine spongiform encephalography has been diagnosed not be used for the manufacture of FDA-regulated products intended for administration to humans. Sourcing of FBS from countries free of bovine spongiform encephalography, including New Zealand or Australia, has become critical when developing cell therapies for clinical use.

In the presence of exogenously added cytokines (TRC-C), the hematopoietic output of the cultures could be enhanced (Tables 1, 2). When the culture medium was changed from medium containing exogenous growth factors conducive to hematopoietic stem cell expansion (TRC-C) to medium containing no exogenous growth factors (TRC), there were diminished numbers of cells of the hematopoietic lineage, such as myeloid cells, and enhanced expansion of cells containing mesenchymal cell markers that correlate with bone scores, that is, CD105+, CD166+, and CD90+. This has a twofold effect in that not only is there an apparent expansion of osteoprogenitor cells, but there is an increase in the relative frequency of osteoprogenitor cells simply from the lack of expansion of hematopoietic cells.

CD13 was used initially to identify myelomonocytic cells within the TRC-C population. CD13 is now also recognized as a mesenchymal cell marker [30, 31]. Dual staining with CD13 and CD90 has shown that 100% of the CD90+ cells in TRCs are also positive for CD13 (supplemental online data). In TRC-C, the majority of the CD13+ cells are myeloid in nature, whereas in the TRC formulation, these cells are mainly mesenchymal. Therefore, this marker is identifying different populations of cells in different TRC formulations. As many markers are expressed on different cell types depending on methods of processing and culturing, it is critical to use multiple surface markers to verify the origin of the cells identified.

As mentioned, TRC culture is distinct from typical MSC cultures in that nonadherent cells were not removed early in culture and cells are not passaged. Performing these steps during culture typically results in a highly purified MSC population with >90% CD90+, CD105+, and CD166+ cells [32, 33]. In contrast, TRC production is a single-step process.

With the presence of accessory hematopoietic or endothelial lineage cells within the TRC product, it is intriguing to speculate that these other cell types may serve to enhance tissue regeneration by providing additional cells or factors that help with revascularization of the developing tissue. In addition, the presence of CD14+ monocyte/macrophage may also serve to affect inflammatory response after tissue injury. The effects of these accessory cell populations are under investigation.

Bone Formation
The results from in vitro osteogenic differentiation and the level of bone formation in vivo followed the same trends. The TRC formulation had the highest levels of calcium deposition and alkaline phosphatase activity in vitro as well as the highest bone scores in vivo. It was noted that the TRC formulation performed similarly to the purified MSC control in these experiments. It was not surprising that the TRC-CAd contained more osteogenic cells than TRC-C since early descriptions of osteogenic potential within bone marrow were ascribed to the highly adherent cell population that formed CFU-F [34].

Correlation
CFU-F and several cell surface markers, including CD90+, CD105+, and CD166+, were found to correlate with bone formation in this in vivo model. The correlation of CD90 with bone formation in vivo is also supported by cell sorting experiments, where all CFU-F and osteogenic potential were found within CD90+ fraction.

Each of these parameters has previously been identified as a measure of MSCs or associated with bone-forming cells. CD105 was originally described as a marker for MSCs [35] and was later identified as endoglin [36]. Antibodies to CD166 or activated lymphocyte cell adhesion molecule were shown to localize to the periosteal region of developing long bones [37] and are also found on MSCs [38]. Anti-CD90 (Thy1) antibody binds to all bone marrow cells selected with the anti-fibroblast antibody D7-FIB [31], and CD90 subpopulations in marrow have been shown to account for a majority of the CFU-F numbers, which correspond to mesenchymal progenitor cells [39]. None of the markers are exclusive to MSCs and bind to other cell types, such as fibroblasts from non-bone marrow sources, endothelial cells, and hematopoietic cells. This is the first study in which these markers have been shown to have a positive correlation with in vivo bone formation. These correlations extend previous observations where the concentration of the marrow prior to injection [40, 41], and in a more recent report, the number of CFU-F progenitors injected into a nonunion repair site, correlated with bone formation [42].

Another marker identified on bone marrow "fibroblasts" is MUC18 (CD146) which, when used as a selection agent, has been shown to account for 90% of the CFU-F units [43]. In this study, the CD146+ cell numbers also correlated with in vivo bone formation, although the assay was run on only two cell preparations. Our preliminary data suggest that >90% of the CD90+ cells in TRCs are coexpressing CD146 (supplemental online data). The antibody STRO-1 is clearly identified with CFU-F formation in marrow [44–46] but was not examined in these studies. However, previous studies showed that cells expressing the STRO-1 antigen were present in the TRC-C product (unpublished data).

The frequency of CD90+ in MSCs is typically over 70% in primary cultures (data not shown) and higher after passaging, compared with 20% in TRC cultures. Although we have shown that CD90 frequency predicts bone forming ability of TRCs (Fig. 3), bone formation in vivo was equivalent between TRCs and MSCs at a loading dose of 5 x 106 cells per milliliter. One possible explanation for this observation is the potential limitation of the assay system. If equivalence between MSCs and TRCs is due to the assay being maximized (the highest bone scores were achieved even at the lowest dose examined), differences may be observed at lower cell loading doses.

If, at lower doses, there is still equivalence between TRCs and MSCs based on total cells loaded, other differences among the culture processes may be responsible for unexpectedly high bone forming capacity of TRCs. Specifically, the presence of accessory cells in the ex vivo culture of TRCs and/or in the in vivo assay may affect bone forming potential of the mixed cell product relative to MSCs, and interactions among CD90+ cells within the TRC cell mixture will be explored further.

Identifying specific cell types that correlate with bone formation would allow the use of cell surface markers recognizing this population to determine the bone-forming potential of a clinical cell preparation prior to use in the patient. The potential to adjust the concentration of these cells by enriching to increase bone forming potential of a cell product, particularly for elderly patients who show lower numbers of these cells, would perhaps enhance clinical outcomes. It is important to note that the correlation of surface markers such as CD90 with bone formation may apply only to TRCs and may not extend to more purified cell populations such as MSCs. Using these results, it may be possible to quantify individual preparations at the time of harvest, calculate the concentration of desired cells, such as CD90+ cells, and potentially adjust the concentration of osteogenic cells to be added to the carrier matrix to be implanted into the patient. Of course, this will require more detailed studies of how implanted TRCs, quantified for these cell surface markers, are able to regenerate bone in an orthotopic location in patients. That type of analysis could be included in clinical trials by running the flow analysis of samples and generating a database of repair outcomes that relate surface marker concentrations to bone healing. In addition, these cell surface markers might also be used to select for the osteoprogenitor cell population prior to use in preparations that are particularly low in cells that correlate with bone formation. This would potentially apply to the elderly population, where some reports show diminished numbers in elderly patients [47, 48], although other studies show no age-related difference [49–51]. Aside from the age-related issue, there is general agreement on individual variation in bone marrow samples [49–54]. Whatever the cause, given the high individual-to-individual variability in osteogenic potential of bone marrow aspirates, the ability to assay for the osteogenic potential of the cell preparation has obvious utility for estimating the clinical dose necessary to effect bone repair.


   SUMMARY
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
Disclosure of Potential...
 Acknowledgments
 References
 
Overall, we have shown that BM MNC can be efficiently expanded ex vivo in a clinically relevant single step automated closed system. The resulting mixed cell products were able to produce bone in vivo at levels comparable to that of more purified MSCs. Flow cytometric analysis of selected cell surface molecules revealed that several mesenchymal markers correlate strongly with in vivo bone scores, thus potentially allowing one to predict bone formation outcomes based on a simple cell surface measurement. Future studies are directed at continued optimization of TRC expansion conditions and understanding the contribution of culture conditions such as single-pass perfusion rates and the presence of accessory cell populations to overall stem and progenitor cell expansion and ability to regenerate tissue.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
References
 
The authors thank Lisa Walsh for her assistance in the animal studies, Brian McEwen for preparation of figures, and Janet Hock for critical review of the manuscript. These studies were supported by a Grant from the NIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) DK074201.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 

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