Clinical-Scale Expansion of a Mixed Population of BoneMarrow-Derived Stem and Progenitor Cells for Potential Use inBone Tissue Regeneration

JAMES E. DENNIS,a KELLY ESTERLY,b AMAD AWADALLAH,a CHRISTOPHER R. PARRISH,b

GREGORY M. POYNTER,b 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

ABSTRACT

Preclinical and clinical studies have demonstrated the abilityof bone marrow derived stem and progenitor cells to regen-erate many tissues, including bone. Methods to expand orenrich progenitors from bone marrow are common; how-ever, these methods include many steps not amenable toclinical use. A closed automated cell production culturesystem was developed for clinical-scale ex vivo production ofbone marrow-derived stem and progenitor cells for hema-topoietic reconstitution. The current study tested the abilityof this bioreactor system to produce progenitor cells, termedtissue repair cells (TRC), possessing osteogenic potential.Three TRC formulations were evaluated: (a) cells culturedwithout exogenous cytokines (TRC); (b) cells cultured withexogenous cytokines (TRC-C); and (c) an adherent subset ofTRC-C (TRC-CAd). Starting human bone marrow mononu-

clear cells (BM MNC) and TRC products were character-ized for the expression of cell surface markers, in vitrocolony forming ability, and in vivo osteogenic potential.Results showed significant expansion of mesenchymal pro-genitors (CD90�, CD105�, and CD166�) in each TRCformulation. In vivo bone formation, measured by histology,was highest in the TRC group, followed by TRC-CAd andTRC-C. The TRC product outperformed starting BM MNCand had equivalent bone forming potential to purified MSCs atthe same cell dose. Post hoc analysis revealed that the presenceof CD90�, CD105�, and CD166� correlated strongly with invivo bone formation scores (r2 > .95). These results demon-strate that this bioreactor system can be used to generate, in asingle step, a population of progenitor cells with potent osteo-genic potential. STEM CELLS 2007;25:2575–2582

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

INTRODUCTION

Efficient tissue repair or regeneration remains a significantchallenge. In the orthopedic field, the use of autologous bonegrafting remains the clinical practice standard of care, althoughit is limited in the availability of graft material and donor sitemorbidity [1]. Another approach to the treatment of nonunionfractures is to harness and expand the inherent osteogenic po-tential of bone marrow cells. Bone marrow was identified as asource of osteoprogenitor cells as early as 1867 [2], and the firstheterotopic transplantation showing bone formation in an ani-mal model was described in 1869 [3] and quickly confirmed inthe following year [4]. Bone marrow has since been shown tocontain cells with multiple mesenchymal lineages [5–13] and,under specific culture conditions, a subset of marrow cells hasbeen shown to express additional phenotypic markers from theectodermal and endodermal lineages [14]. Recent results haveshown that whole bone marrow combined with ceramic carrierscan promote bone repair in animal models [15, 16]. A morerecent development shows the use of the carrier matrix to firstconcentrate the marrow-derived osteoprogenitor cells while italso functions as a scaffold for bone repair [17]. However, noneof these methods using whole bone marrow seek to expand the

osteoprogenitor cell pool and are therefore limited to the numberof osteogenic donor cells that can be harvested from the patient.Patient-specific cell therapy, in many cases, requires large num-bers of cells to replace damaged or diseased tissue. For largebone defects or nonunion fractures, this number may be insuf-ficient for the complete and timely repair and recovery ofskeletal function. Therefore, for many cases, methods may berequired to increase the population of osteogenic cells throughex vivo expansion techniques. Culture-expanded marrow-de-rived mesenchymal stem and progenitor cells have been shownto repair segmental bone defects in animal models [18, 19],including human MSCs implanted into athymic rats [20]. Theexpansion method for mesenchymal stem cells typically in-volves multiple steps in an open process and can take 2–3weeks. For clinical applications, a simple, safe, reproduciblemeans of effectively expanding the osteogenic pool of cellswithin the bone marrow is needed.

We have previously found that single-pass perfusion (SPP)technology results in significant expansion of primary humancells [21–25], particularly increases in stem and progenitorpools, possessing enhanced functional abilities. In SPP, culturemedium is continuously replaced by fresh medium at a slow,controlled rate without disturbance or removal of cells, enablingoptimal exchange of nutrients and metabolic by-products and

Correspondence: James E. Dennis, Ph.D., Department of Orthopaedics, University Hospitals of Cleveland, 6th Floor Hanna Building, 11100Cedar Avenue, Cleveland, Ohio 44106, USA. Telephone: (216) 368-3567; Fax: (216) 368-1332; e-mail: James.Dennis@case.edu ReceivedMarch 26, 2007; accepted for publication May 25, 2007; first published online in STEM CELLS EXPRESS June 21, 2007. ©AlphaMed Press1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2007-0204

TECHNOLOGY DEVELOPMENT

STEM CELLS 2007;25:2575–2582 www.StemCells.com

maintenance of the microenvironment. Specifically, the impor-tance of medium perfusion has been demonstrated not only onthe improved productivity and longevity of BM cultures [21] butalso on the metabolic activity and growth factor production ratesof marrow stromal cells [23].

Application of SPP technology led to the development of anautomated perfused bioreactor system for the clinical-scale ex-pansion 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 optimalculture performance. Bone marrow mononuclear cells (BMMNC) cultured in the bioreactor go through a major change inthe composition of cell populations, resulting in a mixed lineagecell product, termed tissue repair cells (TRCs), that has beenused to generate patient-specific cell therapy for hematopoieticreconstitution [27, 28]. Here, the potential of TRCs is beinginvestigated for its clinical application to bone repair.

TRCs are a mixed cell population containing cell pheno-types normally present in the starting BM MNC, including cellsof hematopoietic, mesenchymal, and endothelial lineages, but atdifferent frequencies. Several modifications of the cell expan-sion and cell harvesting protocols were investigated to identifyculture conditions that would more efficiently enrich for osteo-genic subpopulation of cells. In this study, both in vitro and invivo bone formation results demonstrate that this system is aneffective means for expanding the osteoprogenitor cell popula-tions in marrow, and that several modifications of culture har-vesting techniques or culture components have resulted in en-hanced in vivo bone formation found to be equivalent to morepurified MSC populations. In addition, the frequency of severalcell surface markers correlates with in vivo bone scores.

MATERIALS AND METHODS

Bone Marrow Mononuclear CellsFresh BM MNC from normal donors were purchased from PoieticsInc. (Gaithersburg, Maryland, http://www.lonzabioscience.com)and assessed by flow cytometry, in vitro colony assays, and in vitrodifferentiation assays as described below. The donors ranged in agefrom 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 initiatecultures to generate TRCs. Human bone marrow aspirates wereobtained from University Hospitals of Cleveland with InstitutionalReview Board approval and within NIH guidelines for researchinvolving human subjects. BM MNC were isolated from the bonemarrow aspirate using a Percoll gradient and then used to initiateMSC cultures. In some cases, BM MNC were cryopreserved atapproximately 3.3 � 107 cells per milliliter in cryogenic vials in afinal concentration of 7.5% dimethyl sulfoxide and 20% fetal bo-vine 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°Cand transferred to liquid nitrogen. Vials containing either fresh BMMNC or in some cases frozen BM MNC shipped overnight.

TRC Cell ProductionAfter assessing cell concentration using the Coulter Z2 (BeckmanCoulter, Fullerton, CA, http://www.beckmancoulter.com), BMMNC were inoculated at 2–3 � 108 cells per cell cassette (includesbioreactor and medium reservoir) [26]. Prior to inoculation, biore-actors were primed with long-term bone marrow culture (LTBMC)medium consisting of IMDM, 10% fetal bovine serum, 10% horseserum, 5 �M hydrocortisone, gentamicin sulfate, and vancomycin.The bioreactor is designed such that medium flow rate can beadjusted at any time during the culture process within an operatingrange of 10%–500% volume medium exchange per day dependingon the needs of the culture. The entire volume of the culture area is

approximately 280 ml. For TRC production, LTBMC was passedthrough the bioreactor at a controlled ramped perfusion schedulestarting 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 fromday 8 until harvest on day 12. The cultures were maintained at 37°Cwith 5% CO2 and 20% O2 in the incubator for the duration of theculture. After harvest by trypsinization (0.025% trypsin-EDTA in0.9% sodium chloride), TRCs were shipped overnight at roomtemperature in LTBMC for analysis of in vivo bone formation atCase Western Reserve University. An aliquot of TRCs was alsoanalyzed by flow cytometry and in vitro assays as described below.

Cell Production of TRC Cultured with ExogenousCytokinesBM MNC were cultured as described above for TRCs except thatexogenous growth factors, 25 ng/ml flt3 ligand (Immunex, Seattle,http://immunex.com), 0.1 U/ml erythropoietin (Amgen, ThousandOaks, CA, http://www.amgen.com), and 5 ng/ml PIXY321 (Immu-nex) were present in the culture medium. Again, cells were har-vested by trypsinization and analyzed as described.

Cell Production of an Adherent Subset of TRCCultured with Exogenous CytokinesBM MNC were cultured as described above for TRC cultured withexogenous cytokines (TRC-C). To obtain the plastic adherent sub-population from TRC-C, the nonadherent cells were removed fromthe bioreactor by draining and then discarded. The remaining ad-herent cells in the bioreactor were then trypsinized and analyzed asdescribed. This adherent cell population includes both plastic ad-herent stromal cells and hematopoietic cells closely associated withthe stroma.

MSC CultureBM MNC were placed in 10% FBS in Dulbecco’s modified Eagle’smedium (DMEM) and plated at 1.0 � 107 cells per milliliter in100-mm tissue culture dishes. Medium was exchanged twiceweekly, 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 ofnormal calf serum, centrifugation, and resuspension in serum-freeDMEM.

Flow CytometryStarting BM MNC and the TRC expansion products were washedand resuspended in 1� Dulbecco’s phosphate buffered saline (PBS;Gibco, Grand Island, NY, http://www.invitrogen.com) containing1% bovine serum albumin. Tubes containing 106 cells in 0.5 mlwere stained on ice with various combinations of fluorescentlyconjugated monoclonal antibodies. Viability was determined by7-aminoactinomycin D (7AAD) (Beckman Coulter, Fullerton, CA,http://www.beckmancoulter.com); 7AAD only enters membrane-compromised cells and binds to DNA. Several sets of markerscommonly used to identify mesenchymal cells were used, includ-ing CD90�/CD14�, CD105�/CD166�/CD14�/CD45�, andCD146�. Cells were stained with PC5-conjugated anti-CD90(Thy1) antibodies and fluorescein isothiocyanate (FITC)-conju-gated 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), orCD146-PE (Beckman Coulter). Hematopoietic progenitor cellswere assessed by staining cells with either CD34-PE (BeckmanCoulter) or IgG-PE (control; Beckman Coulter) monoclonal anti-body along with a cocktail of lineage (lin)-specific FITC-conjugatedantibodies: CD3, CD11b, CD15, CD20, and glycophorin-A (Gly-A[CD235a]) (Beckman Coulter). Myeloid progenitors were stainedwith CD13-FITC (Beckman Coulter) along with a lin B cocktail ofPE-conjugated antibodies: CD3, CD11b, CD15, CD20, and Gly-A(CD235a) (Beckman Coulter). Erythroid cells were assessed bystaining with FITC-anti-Gly-A (CD235a). After 15 minutes, cellswere washed and resuspended in 0.5 ml of PBS/bovine serumalbumin (BSA) for analysis on the Epics XL-MCL (Beckman

2576 Clinical-Scale Expansion of Cells with Osteogenic Potential

Coulter) flow cytometer. Results are presented as percent of cellspositive for specific markers after subtracting the value of theisotype 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 AssayCells were plated in 1 ml of LTBMC (see above) in 35-mm tissueculture treated dishes. For BM MNC, 150,000 and 500,000 cellswere plated per dish. For TRCs and MSCs, 100 and 400 cells wereplated per dish. Cultures were maintained for 8 days at 37°C in afully humidified atmosphere of 5% CO2 in air. Colony formingunit-fibroblast (CFU-F) colonies were then stained with Wright-Giemsa, and colonies with greater than 20 cells were counted asCFU-F.

Colony Forming Unit-Granulocyte/MacrophageAssayCells were inoculated in colony assay medium containing 0.9%methylcellulose (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 30% FBS, 1% BSA, 100 �M 2-mercaptoethanol(Sigma), 2 mM L-glutamine (Gibco), 5 ng/ml PIXY321, 5 ng/mlgranulocyte colony-stimulating factor (Amgen), and 10 U/ml eryth-ropoietin. BM MNC were plated at 10,000 and 20,000 cells permilliliter, and TRCs were plated at 1,500 and 3,000 cells permilliliter. Cultures were maintained for 14 days, and colonies withgreater than 50 cells were scored as colony forming unit-granulo-cyte/macrophage (CFU-GM).

Osteogenic Differentiation AssaysCells were cultured for 1–2 weeks in 35-mm dishes containingeither control osteogenic (OS�) medium (DMEM with 10% FBS)or OS� medium (DMEM containing 10% FBS, 100 nM dexameth-asone, 10 mM �-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, ex-pression of alkaline phosphatase (AP), and formation of a mineral-ized matrix by calcium deposition. AP activity present in the dif-ferentiated culture was quantified by measuring the rate ofconversion of p-nitrophenyl phosphate to p-nitrophenol (Sigma) andmeasuring absorbance at 410 nm. Enzyme activity is expressed asnmoles of p-nitrophenol/minute and p-nitrophenol/minute per 106

plated cells. Calcium was quantified following the procedure pro-vided in the Calcium Quantitative Kit (Pointe Scientific Inc., Can-ton, MI, http://pointescientific.com). Briefly, osteogenic cultureswere lysed with 0.5 N HCl, and lysates were collected into micro-centrifuge tubes. After vortexing, each sample was shaken at 500rpm for 4 hours at 4°C. After centrifugation at 1,000g in a micro-

centrifuge, supernatants were collected and assayed for the presenceof calcium by measuring absorbance at 570 nm.

In Vivo Bone FormationTRCs, MSCs, or cell-free DMEM were incubated with fibronectin-coated porous calcium phosphate ceramic blocks (Zimmer, War-saw, IN, http://www.zimmer.com), approximately 27 mm3 in vol-ume, and then implanted subcutaneously into the backs of CB-17severe combined immunodeficient (SCID) mice (CB-17/IcrCrl-SCID-BR; Charles River Laboratories, Wilmington, MA, http://www.criver.com). Cell loading concentrations in these studies were5, 10, 25, and 50 million cells per milliliter. For each experiment,mice were implanted with six sets of ceramics, specifically ceramicsloaded with TRCs at cell concentrations of 5, 10, 25, and 50 millioncells per milliliter, ceramics loaded with MSCs at 5 million cells permilliliter, and ceramics incubated in medium alone. The location ofthe implanted ceramics was systematically changed to avoid anypotential location-specific differences in bone formation. Ceramicimplants were harvested from host SCID mice after 6 weeks, fixedin formalin, decalcified, embedded in paraffin, and serially sec-tioned. Sections were stained with Mallory’s Heidenhain, and visu-ally blinded specimens were scored for presence of bone withinpores. The method of estimating bone formation does not estimatebone volume, rather, the bone score is defined by the number ofceramic pores that contain some bone out of the total number ofpores containing cells on a scale from 0–5, where 0 � no bonedetected, 1 � �0%–20% of pores, 2 � �20%–40%, 3 � �40%–60%, 4 � �60%–80%, and 5 � �80% of pores have bone. This isa subjective bone scoring method that is a modification of a methodused in this laboratory that has been shown to correlate strongly (r2

� 0.77) with direct measurements of bone area percent [29]. Groupswere compared statistically by analysis of variance and Mann-Whitney post hoc test for significance among groups.

RESULTS

Phenotypic Characterization of TRC FormulationsEach preparation of BM MNC was assessed for the expressionof cell surface markers and for the ability to form clonogeniccolonies. The TRC-C formulation, cultured in the presence ofcytokines, was previously shown to reconstitute the hematopoi-etic system in patients and contains both hematopoietic andstromal elements [27, 28]. Variations of this product, includingthe TRC-C adherent layer alone (TRC-CAd) or TRCs generatedwithout exogenous factors (TRC), were also tested. The fre-quencies of selected cell types under different culture and har-vesting conditions are shown in Table 1.

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

Designation PhenotypeBM MNC

(n � 6)TRC-C(n � 5)

TRC-CAd

(n � 3)TRC

(n � 3)

Hematopoietic CD34�

3.4 � 2.0 1.0 � 0.3 1.0 � 0.2 0.7 � 0.2Gly-A

�14.1 � 6.8 12.0 � 6.0 9.9 � 6.5 1.2 � 0.9

CD13�

6.8 � 2.0 34.3 � 7.2 24.7 � 3.7 11.2 � 2.7CD14

�6.4 � 3.1 20.4 � 8.0 28.4 � 5.8 36.4 � 2.4

CFU-GM 0.4 � 0.1 2.1 � 0.9 1.1 � 0.8 0.5 � 0.2Stromal CD90

�0.1 � 0.1 1.5 � 1.4 4.1 � 2.4 18.7 � 2.6

CD105�

1.4 � 0.7 2.2 � 0.9 4.3 � 0.3 13.3 � 1.1CD166

�0.7 � 0.7 1.3 � 1.2 2.9 � 1.6 14.1 � 3.4

CD105�/166�

0.1 � 0.1 0.8 � 0.7 2.2 � 0.3 10.8 � 1.3CD146� (n � 2) 0.5 0.6 n.d. 20.1CFU-F 0.0 � 0.0 0.4 � 0.3 0.6 � 0.3 7.0 � 1.6

Data are presented as mean � SD.Abbreviations: BM MNC, bone marrow mononuclear cells; CFU-F, colony forming unit-fibroblast; CFU-GM, colony formingunit-granulocyte/macrophage; Gly-A, glycophorin-A; n.d., not determined; TRC, tissue repair cell cultured without exogenous cytokines;TRC-C, tissue repair cell cultured with exogenous cytokines; TRC-CAd, adherent subset of tissue repair cells cultured with exogenouscytokines.

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The greatest frequency of CFU-GM, Gly-A�, and CD13�hematopoietic cells is in the TRC-C preparations, followed bythe TRC-CAd, and lastly by the TRC preparation. The mostdramatic difference is observed in the TRC cell product, wherethe frequency of mesenchymal cells, including CFU-F, CD90�,CD146�, and CD105�/166� populations, is dramaticallyhigher than in the other culture conditions. TRCs had more thanfour times the frequency of CD90� cells compared with thenext highest group (TRC-CAd) and approximately five times thefrequency of CD105�/166� cells compared with TRC-CAd.Cell sorting experiments showed that all CFU-F were found inthe 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 assessedby dividing the total number of output cells by the total numberof input cells for each bioreactor. The mean and standard devi-ation for each are shown in Table 2. The TRC-C culture con-ditions result in the greatest expansion of total nucleated cellsand hematopoietic cells such as CFU-GM, CD13�, Gly-A�,CD34�, and CD14� cells, whereas TRC culture conditionspromote the expansion of stromal components (CD90�,CD105�, CD166�, CD146�, and CFU-F) and, at the sametime, a decreased expansion of the hematopoietic elements andtotal nucleated cells. The high degree of variation seen in thefold expansion is primarily a result of high variability of thedonor input frequencies.

In Vitro Osteogenic Potential of TRC FormulationsEach preparation of BM MNC was assessed for its ability todifferentiate along the osteogenic lineage. TRCs were incubatedin OS� (control) or OS� medium for 12 days and assessed forcalcium deposition and alkaline phosphatase activity. Sinceosteogenic potential varies from donor to donor, the results fromone representative experiment comparing TRC formulationsgenerated from the same bone marrow donor are presented inTable 3. TRCs had the highest levels of osteogenic differentia-tion, followed by TRC-CAd and TRC-C. This relative trend hasbeen observed in other experiments comparing either TRCs andTRC-C or TRCs and TRC-CAd. Results from cell sorting ex-periments showed over 500 times more calcium accumulation inthe CD90� fraction (7.30 �g per dish) compared with theCD90� fraction (0.01 �g per dish) in in vitro osteogenic assays.

In Vivo Osteogenic Potential of TRC FormulationsAn in vivo assay for bone formation was used to assess theosteogenic potential of TRCs. In one set of three experiments,TRC-C (cell population cultured with cytokines) was comparedwith TRC-CAd to determine whether osteogenic potential isenriched in the adherent layer. TRC-C contained both nonad-herent and adherent cells, while TRC-CAd is the adherent pop-ulation of TRC-C. For each experiment, a single source of BMMNC was used to inoculate two different bioreactors that wereharvested with or without nonadherent cells. The samples wereimplanted, harvested after 6 weeks, prepared for histologicexamination, and scored for bone formation as described inMaterials and Methods. An example of the histologic results isshown in Figure 1, where 1A and 1B show results from theTRC-CAd group and 1C and 1D from the TRC-C group, bothloaded at 5.0 � 107 cells per milliliter. Many of the pores hereare positive for bone formation and are comparable to the resultsfor MSC, shown in Figure 1E and 1F. Most empty controlceramics were completely negative (Fig. 1G, 1H). The individ-ual results of the bone scores from all three experiments showedthe same general trend of osteogenic response (bone score) after6 weeks in vivo. The results depicted in Figure 1 are shown asexamples of how the in vivo histologic data look and are notintended to be representative of the quantitative results. Forquantitative analysis, the bone score data from all of the sampleswere combined and are shown in tabular form in Table 4. TheTRC-C and TRC-CAd at each cell loading concentration werecompared with each other using the Mann-Whitney U test. Theresults showed significantly higher bone scores for the TRC-CAd over the TRC-C at a cell loading concentration of 5.0 � 107

cells per milliliter (p � .034), and the same trend of the TRC-CAd showing a higher bone score than identically loaded TRC-Csamples was observed for the other three dilutions. The mediumcontrols showed only sporadically positive bone scores, whereasthe 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 andTRC-CAd groups and some differences in the numbers of ce-ramics placed at different locations in the host animal. In total,3 animals were lost out of 36 that were implanted with ceramics.

In a second set of experiments, TRCs, cultured withoutexogenous cytokines and highly enriched for mesenchymalcells, were compared with TRC-Cs and enriched for both he-matopoietic and mesenchymal cells. For each run, a singlesource of BM MNC was used to inoculate two different biore-actors that were cultured either in the presence of growth factors(TRC-C) or the absence of growth factors (TRC). Again, thesamples were implanted, harvested after 6 weeks, prepared forhistologic examination, and scored for bone formation as de-scribed in Materials and Methods. Results in Figure 2 show that,for each donor, the ceramics implanted with TRCs scored high-

Table 2. Fold expansion of cell types

Designation PhenotypeTRC-C(n � 5)

TRC(n � 3)

Hematopoietic Total cells 3.4 � 0.9 0.9 � 0.2CD34 1.4 � 0.6 0.13 � 0.01Gly-A 3.1 � 2.9 0.6 � 0.8CD13 18.1 � 7.5 1.0 � 0.1CD14 13 � 4.9 4.0 � 1.0CFU-GM 13.0 � 7.8 0.6 � 0.3

Stromal CD90 64 � 17 234 � 225CD105 5.9 � 0.5 10 � 1.5CD166 62 � 52 405 � 284CD105/166 45 � 9.0 780 � 343CD146 (n � 2) 5.2 34CFU-F 124 � 53 433 � 155

Data are presented as mean � SD.Abbreviations: CFU-F, colony forming unit-fibroblast; CFU-GM,colony forming unit-granulocyte/macrophage; Gly-A, glycophorin-A; TRC, tissue repair cell cultured without exogenous cytokines;TRC-C, tissue repair cell cultured with exogenous cytokines.

Table 3. In vitro osteogenic potential of TRC

Condition

Calcium (microgramsper dish)

APase activity(nmol/minute per

106 cells)

OS� OS� OS� OS�

TRC-C 0.0 0.0 0.092 0.215TRC-CAd 2.6 93.4 0.498 1.325TRC 23 1,716 2.018 5.215

Abbreviations: OS, osteogenic; TRC, tissue repair cell culturedwithout exogenous cytokines; TRC-C, tissue repair cell culturedwith exogenous cytokines; TRC-CAd, adherent subset of tissuerepair cells cultured with exogenous cytokines.

2578 Clinical-Scale Expansion of Cells with Osteogenic Potential

est for bone formation. Bone scores were equivalent betweenTRCs and MSCs at the same cell-loading dose. A dose responsewas not observed for TRC, as the lowest loading cell dose of5 � 106 cells per milliliter gave maximal bone score. In bothbone marrow samples, there was a low level of positive scoresin the empty controls. Unexpanded BM MNC and all dilutionsof TRC-C had bone scores indistinguishable from those of thenegative 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.

Correlation of Mesenchymal Cells with In VivoBone FormationBecause TRCs showed the highest levels of differentiation invitro and bone formation in vivo, it was hypothesized that thefrequency of cells enriched in TRC preparations might correlatewith in vivo bone scores. To test for this, the in vivo bone scoreswere plotted against the frequency of each of the cell types (Fig.3). Not surprisingly, all of the cells within the stromal ormesenchymal category (CD105�/CD166�, CD90�, andCFU-F) correlated highly with in vivo bone formation. Eventhough the degree of CD105� cell expansion was not dramat-ically different between TRC and TRC-C preparations (less thantwofold difference; Table 2), there was also a clear correlationof CD105� cell numbers with in vivo bone scores. This sug-gests that the overall frequency of specific cell types, rather thantheir fold expansion, seems to be a more important parameter inpredicting the osteogenic potential of the TRC cell product.

DISCUSSION

Overall, these results show that BM MNC can be cultured in anautomated closed-system bioreactor, and the resulting cell prod-ucts possess a diverse range of cell phenotypes with the poten-tial to differentiate down the osteogenic lineage under the rightconditions. The methods of expansion and harvesting of BMMNC-derived cell products lead to unique cell products withdiffering potential for in vivo bone formation. In addition,examination of several cell surface markers revealed a strongcorrelation between the frequency of cell surface markersCD105�, CD166�, and CD90� and in vivo bone formationscores when implanted with a ceramic matrix material.

Ex Vivo ExpansionThe bioreactor system was initially developed for the expansionof hematopoietic stem cells within the context of the marrowsupportive stromal cell microenvironment. The expansion was asingle-step 12-day process within a closed system, and cellswere exposed to a specific medium perfusion schedule estab-lished to enhance stem and progenitor cell output. This processdid not include removal of nonadherent cells early in the cultureprocess as is typical for more purified MSC preparations. Ineach formulation, the same cell phenotypes were present as thestarting BM MNC population, although the frequencies of thesecells were different depending on culture conditions.

The cell culture medium used to generate TRCs containsboth horse and fetal bovine serum. Prior to administering acellular therapy to patients, components such as animal sera aswell as proteolytic reagent compounds must be removed orminimized. Therefore, washing the cell product extensivelyafter harvest is required to reduce or eliminate serum proteinsand trypsin from the cell product. A particular risk with the useof animal derived products is exposure of the cells to infectiousagents. Since 1993, the Food and Drug Administration (FDA)has recommended that bovine-derived material from cattle thathave resided in or originated from countries where bovinespongiform encephalography has been diagnosed not be usedfor the manufacture of FDA-regulated products intended foradministration to humans. Sourcing of FBS from countries freeof bovine spongiform encephalography, including New Zealandor Australia, has become critical when developing cell therapiesfor clinical use.

Figure 1. Histologic sections of ceramics harvested 6 weeks postim-plantation. Ceramics loaded with an adherent subset of tissue repair cellscultured with exogenous cytokines (A, B) and nonadherent tissue repaircells cultured with exogenous cytokines (C, D) at 1.0 � 107 cells permilliliter. The darkly stained regions (arrows) are bone, which is foundadjacent 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.

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

Cell dose (millionsper milliliter)

TRC-C(n � 17)

TRC-CAd

(n � 16)MSC

(n � 33)

5 0.12 � 0.33 0.56 � 0.81 3.5 � 1.010 0.65 � 0.70 1.1 � 0.93 n.d.25 1.8 � 1.2 2.5 � 1.3 n.d.50 2.4 � 1.2 3.5 � 0.82 n.d.

Data are presented as mean � SD. Mean bone score for emptyceramics was 0.06 � 0.24 (n � 33). Differences in n betweenTRC-C and TRC-CAd arose from 3 animals lost afterimplantation.Abbreviations: n.d., not determined; TRC-C, tissue repair cellcultured with exogenous cytokines; TRC-CAd, adherent subset oftissue repair cells cultured with exogenous cytokines.

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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 frommedium containing exogenous growth factors conducive to he-matopoietic stem cell expansion (TRC-C) to medium containingno exogenous growth factors (TRC), there were diminishednumbers of cells of the hematopoietic lineage, such as myeloidcells, and enhanced expansion of cells containing mesenchymalcell markers that correlate with bone scores, that is, CD105�,CD166�, and CD90�. This has a twofold effect in that not onlyis there an apparent expansion of osteoprogenitor cells, but thereis an increase in the relative frequency of osteoprogenitor cellssimply from the lack of expansion of hematopoietic cells.

CD13 was used initially to identify myelomonocytic cellswithin the TRC-C population. CD13 is now also recognized asa mesenchymal cell marker [30, 31]. Dual staining with CD13and CD90 has shown that 100% of the CD90� cells in TRCsare also positive for CD13 (supplemental online data). InTRC-C, the majority of the CD13� cells are myeloid in nature,whereas in the TRC formulation, these cells are mainly mesen-chymal. Therefore, this marker is identifying different popula-tions of cells in different TRC formulations. As many markersare expressed on different cell types depending on methods of

processing and culturing, it is critical to use multiple surfacemarkers to verify the origin of the cells identified.

As mentioned, TRC culture is distinct from typical MSCcultures in that nonadherent cells were not removed early inculture and cells are not passaged. Performing these steps dur-ing culture typically results in a highly purified MSC populationwith �90% CD90�, CD105�, and CD166� cells [32, 33]. Incontrast, TRC production is a single-step process.

With the presence of accessory hematopoietic or endotheliallineage cells within the TRC product, it is intriguing to speculatethat these other cell types may serve to enhance tissue regener-ation by providing additional cells or factors that help withrevascularization of the developing tissue. In addition, the pres-ence of CD14� monocyte/macrophage may also serve to affectinflammatory response after tissue injury. The effects of theseaccessory cell populations are under investigation.

Bone FormationThe results from in vitro osteogenic differentiation and the levelof bone formation in vivo followed the same trends. The TRCformulation had the highest levels of calcium deposition andalkaline phosphatase activity in vitro as well as the highest bonescores in vivo. It was noted that the TRC formulation performedsimilarly to the purified MSC control in these experiments. Itwas not surprising that the TRC-CAd contained more osteogeniccells than TRC-C since early descriptions of osteogenic poten-tial within bone marrow were ascribed to the highly adherentcell population that formed CFU-F [34].

CorrelationCFU-F and several cell surface markers, including CD90�,CD105�, and CD166�, were found to correlate with boneformation in this in vivo model. The correlation of CD90 withbone formation in vivo is also supported by cell sorting exper-iments, where all CFU-F and osteogenic potential were foundwithin CD90� fraction.

Each of these parameters has previously been identified as ameasure of MSCs or associated with bone-forming cells. CD105was originally described as a marker for MSCs [35] and waslater identified as endoglin [36]. Antibodies to CD166 or acti-vated lymphocyte cell adhesion molecule were shown to local-ize to the periosteal region of developing long bones [37] andare also found on MSCs [38]. Anti-CD90 (Thy1) antibody bindsto all bone marrow cells selected with the anti-fibroblast anti-body D7-FIB [31], and CD90 subpopulations in marrow have

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 thelowest cell loading concentration, whereas TRC-C samples from these two donors showed very low scores. Abbreviations: BM MNC, bone marrowmononuclear cells; TRC, tissue repair cell cultured without exogenous cytokines; TRC-C, tissue repair cells cultured with exogenous cytokines.

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

2580 Clinical-Scale Expansion of Cells with Osteogenic Potential

been shown to account for a majority of the CFU-F numbers,which correspond to mesenchymal progenitor cells [39]. Noneof the markers are exclusive to MSCs and bind to other celltypes, such as fibroblasts from non-bone marrow sources, en-dothelial cells, and hematopoietic cells. This is the first study inwhich these markers have been shown to have a positive cor-relation with in vivo bone formation. These correlations extendprevious observations where the concentration of the marrowprior to injection [40, 41], and in a more recent report, thenumber of CFU-F progenitors injected into a nonunion repairsite, correlated with bone formation [42].

Another marker identified on bone marrow “fibroblasts” isMUC18 (CD146) which, when used as a selection agent, has beenshown to account for 90% of the CFU-F units [43]. In this study,the CD146� cell numbers also correlated with in vivo bone for-mation, although the assay was run on only two cell preparations.Our preliminary data suggest that �90% of the CD90� cells inTRCs are coexpressing CD146 (supplemental online data). Theantibody STRO-1 is clearly identified with CFU-F formation inmarrow [44–46] but was not examined in these studies. However,previous studies showed that cells expressing the STRO-1 antigenwere present in the TRC-C product (unpublished data).

The frequency of CD90� in MSCs is typically over 70% inprimary cultures (data not shown) and higher after passaging,compared with 20% in TRC cultures. Although we have shownthat CD90 frequency predicts bone forming ability of TRCs(Fig. 3), bone formation in vivo was equivalent between TRCsand MSCs at a loading dose of 5 � 106 cells per milliliter. Onepossible explanation for this observation is the potential limita-tion of the assay system. If equivalence between MSCs andTRCs is due to the assay being maximized (the highest bonescores were achieved even at the lowest dose examined), dif-ferences may be observed at lower cell loading doses.

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

Identifying specific cell types that correlate with bone for-mation would allow the use of cell surface markers recognizingthis population to determine the bone-forming potential of aclinical cell preparation prior to use in the patient. The potentialto adjust the concentration of these cells by enriching to increasebone forming potential of a cell product, particularly for elderlypatients who show lower numbers of these cells, would perhapsenhance clinical outcomes. It is important to note that thecorrelation of surface markers such as CD90 with bone forma-tion may apply only to TRCs and may not extend to morepurified cell populations such as MSCs. Using these results, itmay be possible to quantify individual preparations at the timeof harvest, calculate the concentration of desired cells, such as

CD90� cells, and potentially adjust the concentration of osteo-genic cells to be added to the carrier matrix to be implanted intothe patient. Of course, this will require more detailed studies ofhow 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 byrunning the flow analysis of samples and generating a databaseof repair outcomes that relate surface marker concentrations tobone healing. In addition, these cell surface markers might alsobe used to select for the osteoprogenitor cell population prior touse in preparations that are particularly low in cells that corre-late with bone formation. This would potentially apply to theelderly population, where some reports show diminished num-bers in elderly patients [47, 48], although other studies show noage-related difference [49–51]. Aside from the age-related is-sue, there is general agreement on individual variation in bonemarrow samples [49–54]. Whatever the cause, given the highindividual-to-individual variability in osteogenic potential ofbone marrow aspirates, the ability to assay for the osteogenicpotential of the cell preparation has obvious utility for estimat-ing the clinical dose necessary to effect bone repair.

SUMMARY

Overall, we have shown that BM MNC can be efficientlyexpanded ex vivo in a clinically relevant single step automatedclosed system. The resulting mixed cell products were able toproduce bone in vivo at levels comparable to that of more purifiedMSCs. Flow cytometric analysis of selected cell surface moleculesrevealed that several mesenchymal markers correlate strongly within vivo bone scores, thus potentially allowing one to predict boneformation outcomes based on a simple cell surface measurement.Future studies are directed at continued optimization of TRC ex-pansion conditions and understanding the contribution of cultureconditions such as single-pass perfusion rates and the presence ofaccessory cell populations to overall stem and progenitor cellexpansion and ability to regenerate tissue.

ACKNOWLEDGMENTS

The authors thank Lisa Walsh for her assistance in the animalstudies, Brian McEwen for preparation of figures, and JanetHock for critical review of the manuscript. These studies weresupported by a Grant from the NIH National Institute of Dia-betes and Digestive and Kidney Diseases (NIDDK) DK074201.

DISCLOSURE OF POTENTIAL CONFLICTS

OF INTEREST

The authors indicate no potential conflicts of interest.

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