Enhanced Engraftment of Mesenchymal Stem Cells in a Cutaneous Wound Model by Cul - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:01 |只看该作者 |倒序浏览 |打印

作者：Carl A. Gregorya, Emigdio Reyesa, Mandolin J. Whitneya, Jeffrey L. Speesb作者单位：aCenter for Gene Therapy, Tulane University Health Sciences Center, New Orleans, Louisiana, USA;bDepartment of Medicine, Cardiovascular Research Institute, University of Vermont, Colchester, Vermont, USA ! B/ U; H& B6 Z  S9 I( Y" {
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      【摘要】
   Human mesenchymal stem cells (hMSCs), also referred to as multipotent stromal cells, are currently being applied in clinical trials for bone diseases, graft versus host disease, and myocardial infarction. However, the standard growth medium for hMSCs contains 10%¨C20% fetal calf serum (FCS), and FCS is strongly immunogenic in both rodents and humans. Previously, we reported that by a sensitive fluorescence-based assay, 7¨C30 mg of internalized FCS is associated with 108 hMSCs, a dosage that will probably be needed for most therapies. We also found that a brief culture in medium containing autologous 20% adult human serum (AHS) or autologous 10% AHS supplemented with growth factors (AHS ) reduced the contamination by more than 99.9%. We have now extensively characterized the culture conditions and shown that hMSC expansion is possible using heterologous 20% AHS or heterologous 10% AHS . The uptake of FCS is an active process that acts to concentrate contamination in the cells even under low serum conditions (2% FCS) but can be actively displaced by incubation of the cells in medium with AHS. Rat MSCs (rMSCs) can be expanded under similar conditions using supplemented heterologous adult rat serum (ARS ). After expansion in FCS, a further 8 days of culture with ARS  significantly improves the viability of the rMSCs in vivo after encapsulation in fibrin followed by subcutaneous implantation in rats. Our results have the potential to dramatically improve cellular and genetic therapies using hMSCs. 6 P" G- A3 B/ C. i
      【关键词】 Adult bone marrow stem cells Stem cell culture Mesenchymal stem cells Immune system Experimental models Ex vivo expansion Engraftment
   INTRODUCTION  @% `9 ]6 y+ J: e, e7 I& K

Human bone marrow-derived mesenchymal stem cells (hMSCs) are currently being applied in clinical trials for bone diseases .6 M& v! O9 h, l( w2 K7 U( J

Previously, we reported that by fluorescence-based assay, 7¨C30 mg of internalized FCS is associated with 108 hMSCs, a dosage that will probably be needed for therapy .

In this study, we have now extensively characterized species-specific culture conditions for hMSCs and rat MSCs (rMSCs). We show that hMSC and rMSC expansion is possible using heterologous species-specific serum for periods sufficient to significantly deplete FCS contamination. The resulting MSCs are phenotypically similar to those grown in standard conditions with FCS. We also demonstrate that uptake of FCS by MSCs is an active process that leads to an intracellular accumulation of bovine antigens even when significantly lower concentrations of FCS are used in the expansion medium (2% FCS). Finally, we describe a rat-based assay of subcutaneous MSC engraftment involving the pre-encapsulation of the cells in a fibrin matrix. With this assay, we show that MSCs exhibit enhanced long-term viability when cultured to remove bovine contaminants. The culture conditions reported in this study provide a means to propagate MSCs and efficiently sustain their survival in immunocompetent models of adult stem cell engraftment. The method has the potential to significantly improve human cellular and genetic therapies using hMSCs.

MATERIALS AND METHODS
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Serum Preparation
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Preparation of human serum was carried out as described previously . Five-hundred milliliters of whole blood was taken from consenting donors who had previously donated bone marrow for preparation of hMSCs. The blood was recovered into 600-ml blood bags (Baxter Fenwal, Deerfield, IL, http://www.baxter.com) in the absence of anticoagulants and allowed to clot for 4 hours at room temperature. The serum (100¨C150 ml) was aspirated from the clot and centrifuged at 500g for 20 minutes. The supernatant was then centrifuged for a further 20 minutes at 2,000g. The cleared serum was incubated at 56¡ãC for 20 minutes to deactivate complement followed by storage at ¨C80¡ãC. Rat serum was prepared as follows: Sprague-Dawley rats were acquired from Charles River Laboratories, Inc. (Wilmington, MA, http://www.criver.com) and allowed to grow to 150¨C250 g. Under anesthesia, the rats were exsanguinated by cardiac puncture with a 10-ml syringe. The blood was allowed to clot for approximately 1 hour and then was centrifuged at 3,000g for 30 minutes. The serum was recovered (approximately 0.5 of the total volume of the draw) and then subjected to further centrifugation for 20 minutes at 3,500g. The cleared serum was incubated at 56¡ãC for 20 minutes to deactivate complement followed by storage at ¨C80¡ãC. Medium containing either the human or the rat serum was filtered through a 0.22-µm membrane before use. It is critical that human or rat serum be prepared and frozen within 6 hours of recovery in the absence of additives.) ]/ G. n" M0 a" H. I! Z
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Fluorescent Serum Preparation9 p" ?' s% l. H7 t
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Fluorescein-conjugated fluorescent FCS (fFCS) and AHS (fAHS) were prepared as described previously . Briefly, the FCS was diafiltered into sodium bicarbonate buffer at pH 9.0, labeled with fluorescein isothiocyanate (FITC), and extensively diafiltered into phosphate-buffered saline (PBS) to remove unreacted FITC. The labeled FCS was then adjusted back to the original protein concentration." q+ }5 }& Q! U5 u
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Tissue Culture0 V6 D7 b* i1 C7 n" K& S6 o) L
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hMSCs were prepared and grown as described previously by the Tulane Health Sciences Center stem cell distribution facility . For fAHS uptake experiments, cells grown in 20% FCS were incubated in medium containing 10% heterologous FITC-labeled human serum supplemented with 10 ng ml¨C1 epidermal growth factor (EGF) and 10 ng ml¨C1 basic fibroblast growth factor (bFGF) for 4 days prior to two PBS washes and digital imaging. For FCS uptake experiments, cells were seeded into 10-cm2 plates (Corning Life Sciences) at 100 cells per cm2 and allowed to grow in complete medium containing 20% FCS for 4 days before replacement with medium containing the indicated concentration of fFCS for 2 days. After two PBS washes, in addition to quantification of fFCS as above (FLX800; BioTek Instruments, Inc.), the cells were visualized by phase-contrast and epifluorescence microscopy (Nikon Eclipse TE200; Nikon, Tokyo, http://www.nikon.com) and documented by digital imaging.

Osteogenic Differentiation and Quantification of Alizarin Red S Staining
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For standard osteogenic differentiation, confluent monolayers of hMSCs were incubated in medium supplemented with 10¨C8 M dexamethasone (Decadron; Merck & Co., Inc., Whitehouse Station, NJ, http://www.merck.com), 50 µg ml¨C1 ascorbic acid, and 5 mM ß-glycerol phosphate (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 40 days with changes of medium every 5 days. Quantification of staining was carried out using a previously described dye extraction procedure ./ m" K3 g( `$ z( ~
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Adipogenic Differentiation and Quantification of Oil Red O Staining

All reagents were purchased from Sigma-Aldrich. Confluent monolayers of hMSCs in six-well plates (10 cm2 per well) were incubated in medium supplemented with 10¨C8 M dexamethasone, 5 x 10¨C8 M isobutylmethylxanthine, and 5 x 10¨C7 M indomethacin. After 30 days, the adipogenic cultures are fixed in 10% formalin for 15 minutes and stained with fresh oil red O solution in 60% (vol/vol) isopropanol in PBS for 20 minutes. Quantification of staining was carried out using a previously described procedure .- e1 Q, Z4 V4 Y

Fibrin Constructs

Blood was recovered from adult Sprague-Dawley rats as described above. The blood was drawn into a 10-ml syringe containing 1.5 ml 200 mM sodium citrate (pH 7.4) (Sigma-Aldrich). The resultant plasma was recovered from the blood by centrifugation at 3,000g for 30 minutes (approximately 0.8 of the total volume of the draw), and then the supernatant was subjected to further centrifugation for 20 minutes at 3,500g. rMSCs were pre-expanded in CCM and then cultured for a further 2 days in the appropriate experimental medium preparation: CCM, osteogenic medium, or adipogenic medium. Cells were recovered by trypsinization, washed in PBS, and then added to the appropriate volume of rat plasma. The plasma/cell suspension was then aliquoted into wells of a 12-well (1 ml) or 24-well (0.5 ml) tissue culture plate (Corning Life Sciences). To initiate clotting, an equal volume of thromboplastin was added (Plastinex; Corning Life Sciences) followed by 0.1 volumes of a 1 M solution of tissue culture grade CaCl2. Clotting was allowed to proceed for 2¨C4 hours, and then the appropriate experimental medium preparation was added to cover the solid construct. The constructs were cultured in CCM for a further 8 days with a change of medium every 2 days. For assays of cell proliferation within the construct, total DNA was quantified by fluorescent labeling of nucleic acids (CyQuant dye; Invitrogen) as described above. DNA recovery was normalized to cell number, and baseline values were acquired from unseeded constructs." \" h; r4 p" N# k4 f0 Q$ S
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Implantation of Constructs Created Under Different Cell Expansion Conditions( K& s5 A# m# k5 {# Y. R6 O: U
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Adult age-matched male Sprague-Dawley rats were separated into three groups of six animals. Before implantation of the constructs, three animals of each group had 0.5 ml of blood drawn from the tail vein under anesthesia. Serum was recovered from the blood as described . Implants containing 300,000 GFP-labeled MSCs pre-expanded in 20% FCS, 20% ARS, or ARS  were cultured in 24-well plates for 7 days at 37¡ãC in humidified air containing 5% CO2. Under anesthesia, the fibrin constructs were implanted subcutaneously between the scapulae. Briefly, the coat was shaved and the skin was sterilized by application of ethanol and iodine. A 15¨C20-mm incision was made longitudinally between the scapulae, and a small cavity was made between the dermis of the skin and the fascia below to accommodate the constructs, which were 10¨C20 mm in diameter. The incision was closed by two or three sutures and then sealed (Vetbond; 3M, St. Paul, MN, http://www.3m.com). After 5 days, the sutures were removed. After 10 days, the procedure was repeated using constructs that contained 106 cells at the same implantation site. Fourteen days thereafter, the animals were placed under anesthesia and euthanized by cardiac exsanguination, and serum was prepared from the blood. The implants with adjacent skin were removed for histology or genomic DNA (gDNA) extraction.
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Immunohistochemistry

Surviving GFP-expressing MSCs were identified by fluorescent antibody labeling. Epifluorescent, DIC (differential interference contrast), and deconvolution microscopy was done using a Leica DM6000B microscope equipped with an automated x, y, z stage and a CCD (charge-coupled device) camera (Leica DFC350 FX; Leica Camera AG, Solms, Germany, http://www.leica.com). Images taken at 0.5-µm intervals were deconvoluted using commercial software (Leica FW4000 and Leica Deblur). Detailed immunohistochemistry methods are available online as supplemental information.& d$ x* c5 x/ ]" a. ?

Polymerase Chain Reaction-Based GFP Assay4 s+ C4 L2 _9 K0 X
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DNA was extracted from the recovered implants with a cell lysis buffer and phenol/chloroform/isoamyl alcohol as described  and gDNA from the transgenic rats themselves were used as standards.

ELISA for Anti-FCS Immunoglobulin G
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The assays were performed as previously described .
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RESULTS4 J' y, E/ H  n4 r: b
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hMSCs Can Be Expanded in Heterologous Human Serum with or Without Growth Factor Supplementation0 c$ k9 Y( U. }3 X  m0 J/ ?) @
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Although 10% FCS is used widely for MSC propagation, we have found that 20% FCS is the optimal concentration for the expansion and maintenance of MSCs, especially at very low densities. For this reason, we have used 20% fetal bovine serum (FBS) as the positive control for all of our assays . To reduce the occurrence of donor-dependent variation, the MSCs (a total of four donors were used in the assays) were chosen at random from an archive containing MSCs that grew well in 20% FCS and exhibited trilineage differentiation into osteoblasts, adipocytes, and chondrocytes. We assumed also that two MSC lines standardized in this manner were sufficient to test the potential of allogenic human serum for the propagation of MSCs for each of the conditions tested. We compared the growth kinetics of hMSCs (n = 2) that were cultured in medium containing 20% FCS, 20% autologous serum, or 20% heterologous serum from one of three different donors (Fig. 1A, 1C).+ P' q1 s0 u5 g5 R+ V  {! n, r
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Figure 1. Proliferation of human MSCs in various media preparations. (A): Proliferation of cultures of MSCs derived from two donors (donor A ) in FCS, FCS , or AHS  prepared with serum from four donors (D  to F ). (C): Final cell recoveries for growth curves in (A). (D): Final cell recoveries for growth curves in (B). Error bars represent SD from six measurements. The values on the y-axis represent the number of recovered cells per tissue culture growth area. Abbreviations: AHS, adult human serum; FCS, fetal calf serum; MSC, mesenchymal stem cell.# S& _! m# r: o5 V
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Cells were recovered from frozen stocks by culture for a brief period in CCM containing FCS. The cells were then transferred to six-well plates at an initial plating density of 100 cells per cm2 and allowed to adhere in the presence of CCM containing FCS for 4 days. The cultures were then transferred to the various media, and the proliferation was measured. MSC growth in 20% autologous or heterologous human serum was not significantly different from cell growth in 20% FCS. Furthermore, in the absence of growth factors, serum from one donor (donor C) demonstrated significantly increased cell growth compared with the other sera tested, including FCS (Fig. 1A, 1C). We then compared the growth kinetics of hMSCs (n = 2) grown in 20% FCS, 10% FCS supplemented with 10 ng ml¨C1 EGF and 10 ng ml¨C1 bFGF (FCS ), 10% autologous serum supplemented with 10 ng ml¨C1 EGF and 10 ng ml¨C1 bFGF, or 10% heterologous serum supplemented with 10 ng ml¨C1 EGF and 10 ng ml¨C1 bFGF (D , E  and F  in Fig. 1B, 1D). For both donors examined, MSCs cultured in supplemented autologous or heterologous serum demonstrated significantly increased growth relative to MSC growth in 20% FCS (Fig. 1B, 1D). MSCs grown in FCS  (10% FCS with growth factor supplements) demonstrated a significant increase in growth compared with cells from the same donor grown in 20% FCS (Fig. 1B, 1D), indicating that the addition of the growth factors was responsible for the observed growth increase. Preliminary data from our laboratory also demonstrated that hMSC lines can be derived from bone marrow aspirates solely expanded in heterologous AHS . It should be noted, however, that due to limitations in serum, the cells were taken only to passage 2." d2 [) i" p$ }8 H' C" r9 V
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Growth of rMSCs Is Inhibited in Heterologous Serum Unless It Is Supplemented with Growth Factors
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We also examined the growth kinetics of rMSCs (n = 2) cultured in 20% FCS, 20% heterologous rat serum, 10% FCS, or 10% heterologous rat serum that was supplemented with 10 ng ml¨C1 EGF and with the lower concentration of 1 ng ml¨C1 bFGF (ARS ) (Fig. 2A, 2B). Unlike hMSCs, rMSCs do not grow well in 10% species-specific serum supplemented with 10 ng ml¨C1 EGF and 10 ng ml¨C1 bFGF but do grow well in 10% species-specific serum supplemented with 10 ng ml¨C1 EGF and 1 ng ml¨C1 bFGF (ARS ) (data not shown). In contrast to the response of hMSCs cultured in 20% heterologous human serum, for both rMSC donors, cell growth in 20% FCS was significantly greater than cell growth in medium containing 20% heterologous rat serum (Fig. 2A, 2B). Cell growth was also significantly increased when rMSCs were cultured in ARS  as compared with growth in 20% heterologous serum or 20% FCS (Fig. 2A, 2B).
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Figure 2. Proliferation of rat MSCs in various media preparations. (A): Proliferation of cultures of MSCs derived from two rat donors (donor A ) in FCS, FCS , or ARS  prepared with serum from 10 rat donors. (B): Final cell recoveries for growth curves in (A). Open bars represent rat donor, and gray shading represents rat donor B. Error bars represent SD (n = 6). The values on the y-axis represent the number of recovered cells per tissue culture growth area. Abbreviations: ARS, adult rat serum; FCS, fetal calf serum; MSC, mesenchymal stem cell.; e: @4 f5 K$ W/ {. [% _

Culture of hMSCs in Medium Containing Heterologous Human Serum Removes FCS Contamination

In a previous study , we demonstrated that medium containing 20% autologous human serum or 10% autologous human serum supplemented with EGF and bFGF could be used to remove FCS contamination from hMSCs that were previously cultured in 20% FCS. To examine the potential for heterologous human serum to remove FCS contamination from hMSCs, we incubated hMSCs in medium containing FITC-labeled 20% FCS (fFCS) for 2 days, washed the cells in PBS, and then cultured the cells for 6 days in serum-free medium (-MEM, Dulbecco's modified essential medium), 20% FCS, FCS , 20% heterologous AHS, or 10% heterologous adult human serum supplemented with 10 ng ml¨C1 EGF and 10 ng ml¨C1 bFGF (AHS ) (n = 2). Assaying fFCS contamination every 2 days, we observed that a significant amount of fFCS contamination was removed in 2 days, and further reduction occurred by 6 days (Fig. 3A, 3B). Incubation in either 20% heterologous AHS or heterologous AHS  resulted in removal of fFCS contamination that was not significantly different from reduction in medium containing unlabeled 20% FCS or FCS  (Fig. 3A, 3B). Similar to incubation in autologous human serum, growth of hMSCs in heterologous human serum for 6 days resulted in removal of 99.99% of FCS contamination (from more than 120 pg per cell to less than 0.5 pg per cell) (Fig. 3A, 3B).

Figure 3. Time course of fFCS depletion in cultures of donor A human mesenchymal stem cells pre-expanded in fFCS and then transferred to various autologous and heterologous media preparations. (A): Six-day time course (left) and rescaled (right) plot of the last 4 days of the time course. (B): Final fFCS contamination level after 6 days of culture. Error bars represent SD (n = 6). The values on the y-axis represent the amount of detected fFCS per recovered cell. Abbreviations: AHS, adult human serum; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; fFCS, fluorescein-conjugated fluorescent fetal calf serum; MEM, minimum essential medium.

Uptake of FCS Contamination Is an Active Process3 s* o# x* G0 h- z1 B6 e4 q3 j

Given that several recent reports have described the growth of MSCs and related cells from bone marrow in medium containing as low as 2% FCS and that 10% FCS is currently the preferred concentration for MSC generation in some clinical trials, we examined whether these culture methods would reduce the levels of FCS contamination to levels that would not generate an immune response (Fig. 4A). MSCs from two human donors were incubated in medium containing 20%, 10%, 5%, or 2% fFCS for 2 days. The cells were then washed with PBS and assayed for fFCS levels using a quantitative fluorescence-based assay . By epifluorescence microscopy, we observed a punctuate pattern of fFCS within the hMSCs, even when cultured in 2% fFCS (Fig. 4A, inset). The uptake of fFCS appeared active given that the decrease in the levels that we measured did not reflect the relative decrease in the percent of fFCS that we included in the culture conditions (Fig. 4A). Incubation of hMSCs in medium containing 2% fFCS for 2 days resulted in the uptake of 20¨C30 pg of FITC-FCS contamination per cell; the injection of 1 x 106 cells in vivo would carry an immunologic dose of 20¨C30 µg, and even 105 cells would carry 2 µg of contaminant, a level that is potentially immunogenic.- g/ t* ]0 `+ l& b7 k  Y

Figure 4. Uptake of fFCS and fAHS  by hMSCs occurs at a range of serum concentrations by an apparently active mechanism. (A): hMSCs were incubated for 2 days in medium containing a range of fFCS concentrations. The cells were then subjected to assays of fFCS uptake. Contamination is disproportionately high even at low levels of serum exposure (inset: fluorescence microscopy of hMSC exposed to 2%  fFCS). Error bars represent SD (n = 6). The values on the y-axis represent the amount of detected fFCS per recovered cell. (B): Epifluorescence microscopy confirms that fAHS  is also taken up by hMSCs. Abbreviations: EGF, epidermal growth factor; fAHS, fluorescein-conjugated fluorescent adult human serum; fFCS, fluorescein-conjugated fluorescent fetal calf serum; FGF, fibroblast growth factor; hMSC, human mesenchymal stem cell.: R. E' _: x- f8 [

To determine whether human serum was taken up from the culture medium in a manner similar to that of FCS, we FITC-labeled adult human serum (fAHS) and cultured hMSCs for 4 days in medium containing 10% heterologous fAHS supplemented with 10 ng ml¨C1 EGF and 10 ng ml¨C1 bFGF. In both low- (50 cells per cm2) or high-density (5,000 cells per cm2) culture conditions (Fig. 4B), hMSCs exhibited a punctuate pattern of fAHS identical to that of fFCS that we observed in previous experiments (Fig. 3B, inset). Furthermore, although we did not assay fAHS levels quantitatively, high-density cultures demonstrated additional staining with fAHS, perhaps due to nonspecific binding of extracellular matrix (Fig. 4B).7 _9 X- f# U( E) D  f% }
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Differentiation Potential of hMSCs Grown in Human Serum

To determine whether growth in human serum could alter the differentiation capacity of hMSCs as compared with growth in FCS, we performed quantitative assays of bone formation (mineralization) and fat formation (lipid production) during periods of 40 and 30 days, respectively. As assayed by Alizarin Red S staining, there was no significant difference in mineral (Ca2 ) deposition between hMSCs grown in 20% AHS versus 20% FCS (Fig. 5A). The addition of 10 ng ml¨C1 EGF and 10 ng ml¨C1 bFGF to culture medium, although stimulating cell growth, resulted in a significant decrease in hMSC mineral production after 20 days for cells grown in either AHS  or FCS  (Fig. 5A)." I3 ~" }. N5 O5 f( ^# Z

Figure 5. Semiquantitative assays of osteogenic and adipogenic differentiation by hMSCs grown in various media preparations. (A): Time course of osteogenic differentiation by hMSCs pre-expanded in FCS, ARS, ARS , or FCS  and then transferred to osteogenic medium with FCS for up to 40 days. Error bars represent SD (n = 6). The values on the y-axis represent the amount of detected Alizarin S stain recovered per 10-cm2 growth area. (B): Time course of adipogenic differentiation by hMSCs pre-expanded in FCS, ARS, ARS , or FCS  and then transferred to adipogenic medium with FCS for up to 35 days. Error bars represent SD (n = 6). The values on the y-axis represent the concentration of detected oil red O stain recovered per 10-cm2 growth area. (C): The shape of constructs containing rMSCs cultured in standard, osteogenic, and adipogenic medium for 7 days. (D): Epifluorescence microscopy (x20 upper, x4 lower) of rMSCs in constructs cultured in the presence of FCS, ARS, or ARS . (E): Proliferation of rMSCs in constructs cultured in FCS, ARS, or ARS . The values on the y-axis represent the number of detected genomes per construct. Error bars are omitted for clarity, but the replicate cell counts (n = 6) deviated by less than 8%. (F): Alizarin Red S staining of histological sections of explanted constructs demonstrating the presence of mineralized tissue in FCS-treated constructs and to a far lesser extent in the ARS -treated constructs. Abbreviations: AHS, adult human serum; ARS, adult rat serum; FBS, fetal bovine serum; FCS, fetal calf serum; h, host tissue; hMSC, human mesenchymal stem cell; HS, human serum; i, implant; OD, optical density; rMSC, rat mesenchymal stem cell.
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In terms of lipid production as assayed by oil red O staining, hMSCs grown in 20% AHS did not differ in their capacity for adipogenesis when compared with cells grown in 20% FCS (Fig. 5B). Alternatively, cells grown in either AHS  or FCS  demonstrated a significant increase in oil red O staining as compared with cells grown without growth factor supplements (Fig. 5B). At the 30-day time point, cells grown in AHS  exhibited significantly greater oil red O staining compared with hMSCs grown in FCS  (Fig. 5B).2 u- {* x7 q3 ?. A$ W: E+ O0 o

In Vivo Tests of Cell Survival in Fibrin Constructs8 w; N; L! Z0 L4 U, t' G
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To test the in vivo viability of the rMSCs grown under various serum conditions, an engraftment assay was employed based on the subcutaneous implantation of the cells encapsulated in coagulated plasma fibrin. MSCs were initially expanded in FCS and then transferred to ARS , ARS, or FCS for a further 5 days. The cells were then recovered by trypsinization and mixed with citrated rat plasma. Upon addition of thromboplastin C and calcium, the plasma coagulated with the cells. The appropriate growth medium was added to the constructs for further culture. Interestingly, in the presence of medium containing FBS, ARS, or ARS , the MSCs caused the construct to shrink and become a dense, compact structure after approximately 4 days (Fig. 5C). When the constructs were cultured in the presence of medium with adipogenic or osteogenic supplements, the constructs became flat, laminar structures that were durable and could be manipulated easily with forceps (Fig. 5C; osteo or adipo). Upon microscopic inspection, it was apparent that the MSCs had spread within the constructs and formed a complex meshwork of processes (Fig. 5D) that formed after a few hours and became progressively complex over 7 days. Extraction of gDNA and quantification confirmed that proliferation occurred within the constructs in all media tested. ARS  medium consistently provided the best proliferative conditions (Fig. 5E).5 C- B. @+ y' W9 ^

To examine the viability of the fibrin constructs in vivo, 20-mm-diameter constructs containing 300,000 GFP rMSCs were synthesized in adipogenic medium and cultured for 7 days in the presence of ARS, ARS , or FCS. Adipogenic medium was chosen to preserve the shape of the construct and to reduce the tendency of the cells to differentiate into mineralizing tissue. The constructs were implanted into Sprague-Dawley rats subcutaneously, between the scapulae. There were no complications resulting from implantation, and the incisions healed after approximately 1 week. After 14 days, new constructs were reimplanted at the same site, this time containing 106 MSCs. Similarly, the constructs were well received by the hosts, and the implantation site healed after approximately 1 week. After 14 days, the implantation sites were removed and subjected to either DNA extraction for evaluation of cell number or immunohistochemistry and histology.2 C) X& w: T3 z9 ?: L- i, e$ p
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When the removed implants were examined macroscopically, the constructs were integrated into the surrounding tissue. Upon histological inspection, the fibrin could be readily visualized. Implants prepared using FCS, and to a lesser extent ARS, often became completely mineralized and stained densely with Alizarin Red S (Fig. 5F). In contrast, all but one of the constructs cultured in ARS  did not mineralize, and in the instance where it was detectable, the maximal degree of calcification was less than 5% of the total volume (Fig. 5F). This is an observation consistent with the ex vivo differentiation data (Fig. 5A) indicating that the ARS  suppressed mineralization of the constructs.
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Immunohistochemical staining of the constructs for GFP confirmed the presence of many rMSCs surrounding the site of the implanted constructs that had been cultured in ARS and ARS  (Fig. 6). The cells cultured in heterologous ARS or ARS  retained their spindle-shaped morphology in vivo, but most had migrated to the periphery of the implant and had integrated into the surrounding host tissue (Fig. 6A¨C6C). In some cases, the fibrin vector was completely clear of rMSCs, and all of them had migrated into the surrounding host tissue (data not shown). Numerous host lymphocytes that stained with an antibody against monocytes and macrophages were identified within the construct but did not appear to be attacking the MSCs grown in ARS or ARS  (Fig. 6D). Because the macrophages/monocytes were detected in all of the constructs from all three groups and the cells were not seen to participate in the destruction of MSCs, it is possible that the monocyte/macrophage response was directed against the construct itself rather than the cells. MSCs that had migrated out of the construct labeled with antibodies against fibronectin (a fibroblast marker) and appeared to be engaged in helping to heal the host wound (Fig. 6E). At other sites outside the construct, migrating transplanted MSCs were negative for fibronectin and appeared instead to be associated with vascular elements (Fig. 6F). In agreement with the PCR ELISA data, only one of the implants from cells cultured in FCS contained cells that were identified by the GFP antibody (data not shown and Fig. 7).
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Figure 6. Engraftment and migration of rMSCs cultured in heterologous species-specific serum and transplanted in fibrin constructs. (A): DIC image of tissue containing MSCs. Fibrin construct is no longer present. (B): Epifluorescence image of tissue shown in (A). GFP rMSCs (red, ALEXA 594 staining) remain engrafted 2 weeks after implantation. (C): GFP rMSCs migrating away from the fibrin construct (arrows). Autofluorescence is turned up to show the construct. (D): Monocyte/macrophage staining of construct localizes host-derived lymphocytes (green, ALEXA 488). Migrating rMSCs (red) are not under immune attack. (E): Some rMSCs (red) that stain for fibronectin, a fibroblast marker (green, ALEXA 488), appear to be engaged in wound repair of the host (arrows, double-positive cells). (F): A portion of the rMSCs (red) that migrate out of the construct are associated with blood vessels (RBCs, yellow autofluorescence) and are negative for fibronectin (green). Abbreviations: Auto, autofluorescence; DIC, differential interference contrast; EPI, epifluorescence; Fibro, fibronectin; GFP, green fluorescent protein; MM, monocyte/macrophage; MSC, mesenchymal stem cell; rMSC, rat mesenchymal stem cell.

Figure 7. Immune responses generated against rMSCs cultured under different conditions and quantification of engraftment. (A): Levels of anti-fFCS IgG in serum before (pre) and after (post) implantation with constructs cultured in FCS, ARS, or ARS  in three animals (A¨CC) per condition. (B): Measurement of recovered eGFP-encoding DNA from explanted constructs cultured in FCS, ARS, or ARS  (PLS on histogram) prior to implantation. The bars represent measurements from single animals, and the y-axis represents the number of eGFP copies detected per microgram of genomic DNA recovered from the implant. The lower plot is a rescaled logarithmic plot of the lower values. Error bars represent SD of three measurements (n = 3). Abbreviations: ARS, adult rat serum; eGFP, enhanced green fluorescent protein; FCS, fetal calf serum; IgG, immunoglobulin G; PLS, ARS .
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Because the probability of graft success in both the ARS and ARS  groups was much higher than in the FCS control groups, but the macrophage response was present in all of the groups tested, it was hypothesized that an IgG-mediated response directed against FCS may account for the phenomenon. To examine this possibility, some of the rats were tested for immunoglobulins directed against contaminating FCS. As expected, rats that received constructs cultured in FCS generated high levels of anti-FCS IgG that probably accounted for the lack of engraftment. Constructs cultured in ARS or ARS  did not elicit the IgG response, despite initial expansion in medium containing FCS (Fig. 7A).
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A PCR ELISA-based quantification assay was employed to measure the degree of rMSC engraftment based on the presence of the eGFP gene. DNA was extracted from the implants, and a portion of the gene encoding 28S ribosomal RNA or eGFP was amplified in the presence of DIG-labeled deoxy nucleotides. The resultant fragments were denatured in alkali and renatured in the presence of a biotinylated oligonucleotide probe coding for a sequence corresponding to an internal stretch of the amplicon. The heteroduplexes were then immobilized on streptavidin-coated microtiter plates, and the DIG-labeled fragments were quantified by immunodetection. Using GFP rat gDNA containing an eGFP-encoding construct as a standard, the number of eGFP copies in the gDNA extracted from the implants could be quantified and related to the total DNA defined by the 28S ribosomal signal. The eGFP-labeled rMSCs could be detected in all of the explants containing constructs cultured in medium containing ARS or ARS , but the degree of engraftment was variable (Fig. 7B), ranging from approximately 10 to 300 copies of eGFP per µg of recovered DNA (ARS n = 5, ARS  n = 6). There was no significant difference between the viability of engrafted rMSCs between the ARS or ARS  groups. In the case of the FCS-treated constructs, the range of engrafted cells was approximately 0.2 to 1 copies of eGFP per µg of recovered DNA for five of six animals (Fig. 7B), measurements that were very close to the threshold of the assay. The remaining animal from this group harbored 240 eGFP copies per µg of recovered DNA, and the reason for such high engraftment compared with the other animals in the FCS group is unclear.
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DISCUSSION
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Our data demonstrate that hMSCs and rMSCs can be expanded efficiently when cultured in medium containing heterologous species-specific serum for a duration that is sufficient to remove most of the FCS contamination that accompanies the MSCs. Although we have not tested MSCs after multiple passages, a brief (up to 12 days) exposure to species-specific serum is sufficient to sustain or even improve proliferative capacity and in vivo viability. hMSCs grown in 20% heterologous human serum grow as well as in 20% FCS, and hMSCs grown in 10% heterologous human serum supplemented with 10 ng ml¨C1 EGF and 10 ng ml¨C1 bFGF expand significantly better than when cultured in 20% FCS. Furthermore, as we demonstrated previously for medium containing autologous species-specific serum .

Transplantation and survival of rMSCs in vivo demonstrates that growth of MSCs in medium containing species-specific serum can improve the probability of graft success probably by virtue of reduced immunogenicity of the cells. Furthermore, ARS  prevents undesirable osteogenic differentiation of the MSCs after implantation. We have not determined whether growth in medium supplemented with EGF and bFGF promotes specification down other cell lineages, although adding the supplements to either FCS or AHS significantly improved the adipogenic potential of the cells. MSCs cultured in ARS and ARS  survived and migrated from the constructs and appeared to engage in host wound repair both as fibronectin-positive fibroblasts and as other unidentified cell types. As expected, host-derived lymphocytes were present at the injury site but did not attack the MSCs grown in heterologous species-specific serum. Early innate immune rejection of implanted cells has been widely documented in the literature, even in immune compromised transplant models, but these observations are generally made when the implanted cells are considerably mismatched with the host (e.g., after xenotransplantation) .9 F( C/ F7 J+ Q) s

In contrast with the constructs grown in species-specific serum, all but one of the constructs cultured in FCS were destroyed by the host. Because the monocyte/macrophage response was universal across all three of the test groups, the greater frequency of graft failure in the FCS test group was probably mediated by an adaptive humoral response given that we detected antibodies against FCS in the blood of all of the animals we assayed that received FCS-cultured cells. Nevertheless, it is not unreasonable to suggest that the increased success of the engrafted constructs containing growth factor-expanded cells may also be due to enhanced viability through nonimmunological mechanisms. For instance, MSCs treated with growth factors and then extensively expanded may be susceptible to cytogenetic aberrancies or telomerase-mediated alterations in cell cycle regulation. Cytogenetic aberrancies, however, are unlikely; in this series of in vivo experiments, the MSCs were at passage 2¨C3 for a maximum of 10 days. Regardless of the presence of growth factors in the medium, the probability of a malignant cytogenetic aberrancy occurring in this study as well as with those aberrancies reported in Spees et al.  were found not to express telomerase upon microarray transcriptome analysis. It is likely, therefore, that the in vivo viability of the MSCs cultured in growth factors may be due to longer inherent telomere lengths but not malignant aberrancies at the level of telomerase or the chromatin.' R  w, T# @" M% \
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SUMMARY* |. Q# {1 B4 y1 ~. M$ S* s5 k
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The extent of the migration of the cells and their in vivo longevity is still under investigation, but these preliminary data suggest that fibrin vectors and species-specific culture conditions may provide an efficient strategy of MSC administration that maximizes viability and efficacy.0 G+ l/ b5 t# n6 s  @/ L
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DISCLOSURES2 S: b0 `( K( D6 ?/ V; N* Y9 K
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The authors indicate no potential conflicts of interest.. b' F9 W% I- A

ACKNOWLEDGMENTS# ^- u9 g% j8 @' g. I
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C.A.G. and J.L.S. contributed equally to this work. This work was funded by the National Institutes of Health (1P01HL075161 and HL077570-01), The Louisiana Gene Therapy Consortium, and HCA, The Health Care Company.
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