- Email: [email protected]
Immunophenotype characterization of rat mesenchymal stromal cells
Cytotherapy (2008) Vol. 10, No. 3, 243253
Immunophenotype characterization of rat mesenchymal stromal cells MT Harting1,2, F Jimenez1,2, S Pati3,4, J Baumgartner1 and CS Cox1,2 1
Department of Pediatric Surgery and 2The Trauma Research Center, University of Texas Medical School at Houston, 3Department of Neurobiology and Anatomy, University of Texas Medical School at Houston, and The Vivian L. Smith Center for Neurologic Research, and 4 Department of Physical Medicine and Rehabilitation, Baylor College of Medicine, Houston, Texas, USA
Background Mesenchymal stromal cells (MSC) have shown diverse therapeutic potential. While characterization of human and mouse MSC has seen significant advances, rat bone marrow-derived MSC (rBM-MSC) remain under-characterized. We detail the isolation, expansion, differentiation, and detailed immunocharacterization of rBM-MSC. Methods Rat MSC were isolated and expanded in multipotent adult progenitor cell (MAPC) media, and cell-surface marker expression through 10 passages was used to characterize the population and multipotency was confirmed via differentation. Results By passage 3, rBM-MSC were found to be CD11b , CD45 , CD29 , CD49e , CD73 , CD90 , CD105 and Stro-1 ,
Introduction Mesenchymal stromal cells (MSC) have shown great promise as therapeutic agents in various fields of study [15]. However, varied isolation and expansion techniques, along with diverse methods of cell characterization, have yielded ill-defined and incomparable cellular populations. While characterization of human and mouse MSC has seen significant advances in the last few years [610], characterization of rat bone marrow-derived MSC (rBMMSC) has lagged behind. Rats serve as critical models in numerous diseases where cellular therapy is being studied. As these results are translated to clinical trials, ensuring consistent cellular populations is vital. Therefore, we
without the use of cell sorting. Media selection was responsible for the isolation of a nearly homogeneous population of rBM-MSC. The rBM-MSC immunophenotype changed by passage 10, showing decreases in CD73, CD105 and Stro-1 expression. Discussion Detailed characterization of cell populations facilitates accurate and reproducible cell therapy investigation. Given the expanding body of research involving rBM-MSC, these results advance our ability to compare rBM-MSC populations. Keywords cell therapy, immunocharacterization, marker expression, mesenchymal stromal cell, rodent, stem cell.
sought to isolate, expand and characterize the immunophenotype of rBM-MSC. Since the early 1970s MSC have been defined by their ability to adhere to plastic and proliferate [11]. Nearly 40 years later, this property remains the key step in the isolation of MSC. More recently, MSC have been defined further by their ability to differentiate down multiple lineages (most commonly adipocytes, chondrocytes and osteocytes) [6,12], including reports of differentiation into neural [13,14] and hepatic [15], among other, cell types. Cells similar to MSC, known as multipotent adult progenitor cells (MAPC), have been reported to differentiate down all three germ cell lines [16]. Others have
Correspondence to: Charles S. Cox, Jr, MD, Department of Pediatric Surgery, University of Texas Medical School at Houston, 6431 Fannin St, MSB 5.254, Houston, TX 77030, USA. E-mail: [email protected] – 2008 ISCT
DOI: 10.1080/14653240801950000
244
MT Harting et al.
defined MSC based on cell surface marker expression or lack thereof [7,8]. Most of the defining properties, markers and actions that characterize rBM-MSC were originally identified while using human and mouse cell lines [6,10,16]. Many of the same characteristics have been shown to transcend species, however some may not. We detail our methods for isolating rBM-MSC. We do not require magnetic-activated cell sorting (MACS) or fluorescent-activated cell sorting (FACS) to isolate our population of rBM-MSC. We follow three cell lines through to passage 10, characterizing the following cell-surface markers over time via flow cytometry: CD45, CD11b, CD29, CD49e, CD73, CD90, CD105 and Stro-1. A near homogeneous population of rBM-MSC is seen by passage 3 (14 days). By passage 8 or 9 (c. 1 month), cells develop suboptimal growth patterns and altered cell-surface marker expression. The rBM-MSC were differentiated to adipocytes, chondrocytes and osteocytes, confirming their multipotency.
Methods Cell isolation from BM SpragueDawley rats (200225 g) were purchased from Harlan Sprague Dawley (Indianapolis, IN, USA) for use in this study. The animals were housed on a 12-h light/dark cycle with ad libitum access to food and water. All protocols involving the use of animals were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (protocol HSC-AWC06038). Immediately after killing, the muscle was dissected from the femurs and tibiae, leaving isolated bone. The tips of the bones were shaved off with a Rongeur. Each bone was flushed with c. 10 ml of Medium 199 (Gibco, Carlsbad, CA, USA), leaving a cell suspension. After repetitive flushing (1015 times) through an 18-gauge needle, the cell suspension was filtered through a 40-mm nylon cell strainer (BD Falcon, Bedford, MA, USA). The cell suspension was then centrifuged for 6 min at 800 g. Cells were counted and plated in 6-well plates (Nunc, Rochester, NY, USA) coated with fibronectin (Sigma, St. Louis, MO, USA; 100 ng/mL in 1 PBS) at c. 106 cells/cm2 (c. 107 cells/well) in 2 mL media/well. Warm media (1 mL) were added after 48 h. After 72 h, all media were removed, the wells gently washed with 1 PBS, and refed with fresh, warm media. Media were replaced every 48 h for another 46 days. All
cells were then separated from the plastic (passage), using 0.05% trypsin EDTA (Cellgro, Manassas, VA, USA), every 4872 h, through to passage 10. See Figure 1 for an overview of cell isolation and expansion. Cells were maintained in a rat media mixture as described previously [17]. The rat media contains 60% low-glucose Dulbecco’s modified Eagle media (DMEM; Gibco), 40% MCDB-201 (Sigma), 1 insulin-transferrinselenium (ITS; Sigma), 1 linoleic acid bovine serum albumin (LA-BSA; Sigma), 10 9 m dexamethasone (Sigma), 10 4 m ascorbic acid 3-phosphate (Sigma), 100 U penicillin, 1000 U streptomycin (Gibco), 2% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 10 ng/ mL human platelet-derived growth factor (R&D Systems, Minneapolis, MN, USA), 10 ng/mL mouse epidermal growth factor (Sigma) and 1000 U/mL mouse leukemia inhibitory factor (Chemicon, Temecula, CA, USA). Cells isolated and expanded in the above media (MAPC media) were compared with cells isolated and expanded in basic media consisting of DMEM (low glucose; Gibco),supplemented with 10% FBS (HyClone) and penicillin/streptomycin (Gibco).
Flow cytometric characterization Flow cytometry analysis was performed using a BD LSR II (BD Biosciences, San Jose, CA, USA). Cells were collected from three SpragueDawley rats and flow cytometric analysis performed at passage zero (P 0 or BM) and P 1, 2, 3, 4, 5 and 10. For each passage, cells were detached with trypsin (as above) and labeled with a fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated primary antibody (Ab) or purified primary Ab, followed by a conjugated secondary Ab. The following cell-surface markers were characterized at each passage: CD11b, CD45, CD49e, CD73, CD90 (all from BD Biosciences), CD105 (Santa Cruz, Santa Cruz, CA, USA), CD29 (BioLegend, San Diego, CA, USA) and Stro-1 Bone Marrow Isolation
Remove non-adherent cells
Day 0
Day 3
1st Passage (P=1)
P=2
P=3
P=4
P=5
Day 7
Day 9
Day 11
Day 13
Day 15
P=10
Day 25
Flow Cytometric Analysis CD 11b CD 45 CD 29 CD 49e CD 73 CD 90 CD 105 Stro-1
Figure 1. Overview of cell isolation, expansion and immunophenotyping.
Immunophenotype characterization of rat MSC
(Zymed (now Invitrogen, Carlsbad, CA); Jackson ImmunoResearch, West Grove, PA, USA). Each Ab is specific to rat markers. Secondary Ab were purchased from BD Biosciences, Zymed or Jackson ImmunoResearch. Standard labeling protocols and the manufacturers’ suggestions were followed. Cells were suspended at 106 cells/mL and incubated with primary Ab (1:100, v/v) for 30 min at room temperature (RT). If not primarily conjugated, the cells were subsequently incubated with secondary Ab (1:100, v/v) for 30 min at RT. For the CD105 Ab, cells were fixed with 4% paraformaldehyde (PFA) for 30 min prior to incubation with primary Ab. The MSC population was identified based on forward/side scatter (FSC/SSC) properties. This population was gated to collect a maximum of 10 000 events.
Differentiation media/induction protocol/ staining Prior to differentiation, rBM-MSC were plated in rat media at 104 cells/cm2 in 12-well plates. The cells were allowed to adhere to the plastic and were near confluence after 2448 h. Undifferentiated rBM-MSC were stained using the identical protocols for Oil Red O (adipocytes), Alcian Blue (chondrocytes) and Alizarin Red S (osteocytes), as described below. These served as negative controls. For adipogenic differentiation, cells were plated in the presence of alpha MEM medium (Invitrogen, Carlsbad, CA, USA) containing 12.5% horse serum (Invitrogen), 12.5% FBS (HyClone), penicillin/streptomycin (50 U/ mL; Gibco), b-mercaptoethanol (10 4 m; Sigma) and hydrocortisone (10 4 m; Stem Cell Technologies, Vancouver, BC, Canada). The media were changed completely every 4872 h. The cells were allowed to differentiate for 1428 days. Characterization of adipocytes was performed by Oil Red O staining (Diagnostic Biosystems, Pleasanton, CA, USA) of the intracellular lipid-rich vacuoles. After fixation with 4% PFA for 30 min at RT, cells were washed and incubated in Oil Red O solution for 50 min at RT. After washing, cells were counterstained with Mayer’s hematoxylin for 5 min at RT. Cells were immediately imaged using inverted microscopy (Nikon, Melville, NY, USA). For chondrogenic differentiation, cells were cultured at 3 105 cells/mL in serum-free chemically defined medium consisting of DMEM (high-glucose; Invitrogen), 1 insulin-transferrin-selenium (Sigma), 1 LA-BSA
245
(Sigma), ascorbate 2-phosphate (50 mg/mL; Sigma), dexamethasone (100 nm; Sigma) and 10 ng/mL hTGF-b1 (R&D Systems). The media were changed completely every 4872 h. The cells were allowed to differentiate for 1428 days. Characterization of chondrocytes was performed by Alcian Blue staining (Richard Allen Scientific, Kalamazoo, MI, USA) of the proteoglycan-rich cartilage matrix. After fixation with 4% PFA for 30 min at RT, cells were washed and incubated in Alcian Blue solution for 30 min at RT. The staining solution was removed thoroughly via 34 distilled water washes. Cells were immediately imaged using inverted microscopy (Nikon). For osteocyte differentiation, cells were plated in the presence of osteogenic differentiation media (Lonza, Walkersville, MD, USA) that contained basal medium, dexamethasone, ascorbate, mesenchymal cell growth supplement, l-glutamine, penicillin/streptomycin and b-glycerophosphate. The media were prepared according to the manufacturer’s instructions. The media were changed completely every 4872 h. The cells were allowed to differentiate for 1428 days. Characterization of osteocytes was performed by Alizarin Red S indicator staining (Ricca Chemical Company, Arlington, TX, USA) for calcium formation. After fixation with 70% ice-cold ethanol for 1 h at RT, cells were washed and incubated in Alizarin Red S solution for 30 min at RT. The staining solution was removed thoroughly via 34 distilled water washes. Cells were immediately imaged using inverted microscopy (Nikon).
Analysis Flow cytometry data were collected using a BD LSR II and initial statistical analysis was performed using BD FACS Diva Software (BD Biosciences; Microsoft Excel (Microsoft, Redmord, WA, USA)). The data were further analyzed and graph overlays created using FlowJo software (version 7.2.1; Tree Star Inc., Ashland, OR, USA). Graphs of marker percentage versus passage number were generated using Microsoft Excel (Excel Professional 2003). All numbers are expressed as mean9SEM.
Results We initially identified rBM-MSC after 46 days of culture in media (Figure 2A). An additional few days (35 days, with media changes every 48 h) allowed the proliferating rBM-MSC to expand clonally without overcrowding. Rat MSC form a characteristic triangular or ‘fibroblastic’
246
MT Harting et al.
Figure 2. (A) Heterogeneous population of BM cells after 56 days in culture. MSC are distinguished by their characteristic spindle or fibroblastic shape when adherent to plastic. (B) A nearly homogeneous population of MSC (passage 3).
appearance when adherent to plastic (Figure 2B). Over subsequent passages the rBM-MSC maintained their morphological appearance. We found cell doubling times to range between 12 and 48 h, dependent upon passage number. After 910 passages a decreased growth rate and larger cell mass were noted. The newly isolated rat BM contained a mixture of cells identifiable by flow cytometry (Figure 3A, B). Cells found in the area where rBM-MSC are identified in future passages were largely positive for CD90, CD29, CD49e, CD73, CD105 and Stro-1; however, they were also largely positive for CD11b and CD45 (Figure 3B, C). Cells located in the mononuclear cell and lymphocyte areas were found to express CD90 (and the other positive markers above) while lacking CD11b (and CD45) expression (Figure 3B, C).
In the BM populations, we found some variability between different rats in the expression of the eight markers studied (Figure 4). In general, MSC have been defined as a population negative for CD11b and CD45. CD11b and CD45 were found on 34.790.74% and 75.69 8.58%, respectively, of newly isolated rat BM cells. Markers that are reported to be present on MSC were found on relatively small subsets of rat BM cells (Figure 4). CD73 and CD105 were found on less than 20% of cells, while other markers were noted on as many as 40% of the total cell population. Over the first three passages, rBM-MSC emerged from a heterogeneous population to a more homogeneous population (Figure 5AE). In the BM, cells positive for CD90 and negative for CD11b could be found in the mononuclear cell area (Figure 5A). After the first passage, few CD90 CD11b cells could be found (Figure 5B). By the second passage, the population expressing markers characteristic of MSC began to emerge (Figure 5C). An expanded population of CD90 CD11b cells could be seen by passages 3 and 4 (Figure 5D, E). Over the first three cell passages, CD11b and CD45 cells declined rapidly, each falling to less than 10% by passage 2, around 5% by passage 3 and less than 2% by passage 4. The percentage of cells expressing CD29, CD49e and CD90 rapidly increased to 90% by passage 2 and remained above 99% after passage 4. The other markers showed slightly increased variability. Markers CD73, CD105 and Stro-1 reached peak expression between passages 3 and 6. All markers showed somewhat decreased expression by passage 10. Cell marker expression over 10 passages is shown in Figure 4. Histogram overlays of each marker over the first three passages (until a near homogeneous population existed) are shown in Figure 6. Given previous reports of the inability to isolate a pure population of MSC, exclusive of hematopoietic stem cells or macrophages, in culture without cell sorting [8], we compared isolation and expansion in the MAPC media with isolation and expansion in a basic media consisting of DMEM, FBS and penicillin/streptomycin. We found that 2030% CD11b and CD45 cells remained after isolation and three passages in the basic media. Cells isolated and expanded through three passages in MAPC media, however, were found to be CD11b and CD45 (Figure 7).
Immunophenotype characterization of rat MSC
247
Figure 3. BM cell scatter plot (A) revealing the characteristic cellular populations (B). Although rMSC will eventually appear in the MSC area, a small fraction of cells in the monocyte or lymphocyte area are CD90 CD11b (C).
248
MT Harting et al.
CD 11b
60 40 20 0
0
2
4 6 Cell Passage number
8
10
CD 29 100 80 60 40 20 0
0
2
4 6 Cell Passage number
8
10
CD 73 100 80 60 40 20 0
0
2
4 6 Cell Passage number
8
10
CD 105 100 80 60 40 20 0
0
2
4 6 Cell Passage number
8
10
Percentage Positive for marker (%)
80
Percentage Positive for marker (%)
100
Percentage Positive for marker (%)
CD 45
Percentage Positive for marker (%)
Percentage Positive for marker (%)
Percentage Positive for marker (%)
Percentage Positive for marker (%)
Percentage Positive for marker (%)
D
100 80 60 40 20 0
0
2
4 6 Cell Passage number
8
10
8
10
8
10
8
10
CD 49e 100 80 60 40 20 0
0
2
4 6 Cell Passage number CD 90
100 80 60 40 20 0
0
2
4 6 Cell Passage number Stro-1
100 80 60 40 20 0
0
2
4 6 Cell Passage number
Figure 4. Cell-marker expression percentage through 10 passages for the eight surface markers analyzed. Cells from passage 3 display cell-surface marker expression characteristic of MSC. By passage 10, there is a decline in marker expression of CD73, CD105 and Stro-1.
Differentiation of the rBM-MSC to adipocytes, chondrocytes and osteocytes led to expected changes in cell morphology and staining characteristics, confirming their multipotency. Staining of passage 3 rBM-MSC controls with Oil Red O, Alcian Blue and Alizarin Red S identified no typical characteristics of adipocytes, chondrocytes or osteocytes, respectively (Figure 8A, D, G). After being exposed to the differentiation media, the cells developed the staining characteristics expected for adipocytes (Figure 8B, C), chondrocytes (Figure 8E, F) and osteocytes (Figure 8H, I). We found that c. 6070% of cells stained positive with Oil Red O (adipocytes) and that the differentiated adipocytes remained adherent to plastic. The chondrocytes gathered into large spheres that stained deeply positive
with Alcian Blue, consistent with human/mouse differentiation. Among individual chondrocytes, c. 5060% of the cells stained positive with Alcian Blue. Osteogenic differentiation led to a rapid phenotypic change toward cuboidal morphology and some delamination from the culture surface. In areas where cells remained adherent, the entire differentiated cell mass stained bright orange after staining with Alizarin Red S.
Discussion We have isolated and characterized a population of rBMMSC. We found that a nearly pure population of rBMMSC was achievable by passage 3, without the use of MACS or FACS. The cells continued to expand well and
Immunophenotype characterization of rat MSC
249
Figure 5. (A) Expansion of the rMSC population over the first four passages. The BM contains relatively few CD90 CD11b cells. (B) After passage 1, the number of CD90 CD11b cells remains relatively small. (C) By passage 2, the population of rMSC reaches 5075% of the total cellular population. The CD90 CD11b cellular population is 98% by passage 3 (D) and remains homogeneous in passage 4 (E).
250
MT Harting et al.
Figure 6. Overlays for all cell markers analyzed over three passages. The first column shows the FSC/SSC of the complete population, with a box around the gated cell population.
maintained a consistent phenotype, through passages 78. By passage 8 or 9, rBM-MSC had developed suboptimal growth patterns and immunophenotype changes. Given proper media conditions, the rBM-MSC were induced to differentiate down adipogenic, chondrogenic and osteogenic phenotypes, as evidenced by the development of specific staining characteristics.
The characterization of MSC has seen significant progress over the last decade. Human MSC isolated from the BM were initially and thoroughly characterized by Pittenger et al. in 1999 [6]. Human MSC isolated from other locations were subsequently characterized [18]. Murine MSC characterization followed shortly thereafter [10,19]. Rat MSC have been somewhat characterized [20
Figure 7. Overlay showing cell marker expression (passage 3) with MAPC media and basic media. After three passages in basic media, a significant cell population continues to express CD11b and CD45, while cells expanded in MAPC media are CD11b and CD45 negative.
Immunophenotype characterization of rat MSC
251
Figure 8. Rat MSC differentiation to adipocytes (AC), chondrocytes (DF) and osteocytes (GI). (A) Undifferentiated rMSC stained with Oil Red O at 10 (control); (B) adipocyte-differentiated rMSC stained with Oil Red O at 10 ; (C) adipocyte-differentiated rMSC stained with Oil Red O at 30 ; (D) undifferentiated rMSC stained with Alcian Blue at 4 (control); (E) chondrocyte-differentiated rMSC (spheroid) stained with Alcian Blue at 10 ; (F) chondrocyte-differentiated rMSC stained with Alcian Blue at 20 ; (G) undifferentiated rMSC stained with Alizarin Red S indicator at 4 (control); (H) osteocyte-differentiated rMSC stained with Alizarin Red S indicator at 4 ; and (I) osteocytedifferentiated rMSC stained with Alizarin Red S indicator at 10 .
22], although many seem to assume that they share similar characteristics to murine MSC. Through rigorous work by many groups, human/murine MSC have been described as generally positive or negative for the following markers (not species specific): CD11b , CD14 , CD34 , CD45 , CD13 , CD29, CD44, CD49e , CD71 , CD73, CD90 , CD105 , CD106 , CD120a , CD124 and Stro-1 . Given the limited availability of commercial Ab specific to the rat, we identified available Ab for these markers specific to rat cells. A complete understanding of the immunophenotype of species-specific cell populations is a key part of cellular
characterization. In 2006, Dominici et al. [23] outlined minimal criteria for defining human MSC. The criteria were: (1) adherence to plastic in standard culture conditions; (2) expression of CD105, CD73 and CD90 combined with a lack of expression of CD45, CD34 and CD14 or CD11b and CD79 or CD19 and HLA-DR surface molecules; and (3) differentiation into osteoblasts, adipocytes and chondroblasts in vitro. Cell characterization by surface molecule expression is rapid, highly specific and requires a small cell number. Immunophenotyping is likely to become the standard for cell characterization and comparison of cellular populations.
252
MT Harting et al.
Many reports of MSC isolation and characterization discuss the importance of cell sorting in order to develop a pure population of cells [7,8,12,16,24]. They propose the use of MACS or FACS in order to eliminate other hematopoetic or non-stem cells. They feel that without such sorting a heterogeneous population of cells, including hematopoetic stem cells and macrophages, will remain. Although non-MSC resilience is seen with rBM-MSC isolation, it has been particularly problematic when isolating mouse MSC. Schrepfer et al. [8] have shown that, in their cultures, macrophages are not effectively eliminated until after passage 10. Our data indicate that sorting may not be necessary to develop a homogeneous (95%) population of rBM-MSC. We show that MAPC media is probably responsible for this phenomenon. We found that 95% of the cells were CD11b , CD45 , CD29, CD49e, CD73, CD90, CD105 and Stro-1 by the third passage. Such a pattern of cell-surface marker expression is congruent with the surface markers previously characterized for human and mouse MSC. Given our findings, we make the following recommendations for rMSC isolation and expansion. (1) Rat BMMSC in passages 38 show the most uniform immunophenotypic characteristics (earlier passages show a mixture of phenotypic surface molecules and later passages experience phenotypic changes). (2) MACS and/or FACS may be unnecessary to achieve a nearly homogeneous population of rBM-MSC. Isolation and expansion in MAPC media leads to a more homogeneous population over few passages compared with the commonly used basic media. (3) Characterization of high-passage ( 810 passages) rBM-MSC by CD90 , CD29, CD49e , CD45 and/or CD11b alone may not accurately reflect the immunophenotype. (4) Rat BM-MSC were found to be CD11b , CD45 , CD29 , CD49e , CD73, CD90, CD105 and Stro-1 by passage 3; immunophenotypic characterization may become the standard for identifying and comparing rBM-MSC, given the high specificity, low cell number requirement and speed.
Acknowledgements We acknowledge The University of Minnesota Stem Cell Institute (Minneapolis, MN, USA), specifically Yuehua Jiang and Catherine Verfaillie (now in Belgium), for the generous opportunity to train in their NIH sponsored
program. Supported by NIH grant T32 GM008792-06 (M. T. Harting).
References 1 Oreffo RO, Cooper C, Mason C, Clements M. Mesenchymal stem cells: lineage, plasticity, and skeletal therapeutic potential. Stem Cell Rev 2005;1:16978. 2 Bussolati B, Camussi G. Adult stem cells and renal repair. J Nephrol 2006;19:7069. 3 Khakoo AY, Pati S, Anderson SA et al. Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi’s sarcoma. J Exp Med 2006;203:123547. 4 Leo AJ, Grande DA. Mesenchymal stem cells in tissue engineering. Cells Tissues Organs 2006;183:11222. 5 Pittenger MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res 2004;95:920. 6 Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:1437. 7 Gang EJ, Bosnakovski D, Figueiredo CA et al. Ssea-4 identifies mesenchymal stem cells from bone marrow. Blood 2007;109: 174351. 8 Schrepfer S, Deuse T, Lange C et al. Simplified protocol to isolate, purify, and culture expand mesenchymal stem cells. Stem Cells Dev 2007;16:1057. 9 Martinez C, Hofmann TJ, Marino R et al. Human bone marrow mesenchymal stromal cells express the neural ganglioside gd2: a novel surface marker for the identification of MSCs. Blood 2007;109:42458. 10 Tropel P, Noel D, Platet N et al. Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow. Exp Cell Res 2004;295:395406. 11 Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guineapig bone marrow and spleen cells. Cell Tissue Kinet 1970;3:393 403. 12 Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:714. 13 Brazelton TR, Rossi FM, Keshet GI, Blau HM. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000;290:17759. 14 Mezey E, Chandross KJ, Harta G et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:177982. 15 Petersen BE, Bowen WC, Patrene KD et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:116870. 16 Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:419. 17 Breyer A, Estharabadi N, Oki M et al. Multipotent adult progenitor cell isolation and culture procedures. Exp Hematol 2006;34:1596601.
Immunophenotype characterization of rat MSC
18 Mitchell JB, McIntosh K, Zvonic S et al. Immunophenotype of human adipose-derived cells: temporal changes in stromalassociated and stem cell-associated markers. Stem Cells 2006;24: 37685. 19 Meirelles Lda S, Nardi NB. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol 2003;123:70211. 20 Zangi L, Rivkin R, Kassis I et al. High-yield isolation, expansion, and differentiation of rat bone marrow-derived mesenchymal stem cells with fibrin microbeads. Tissue Eng 2006;12:234354. 21 Gindraux F, Selmani Z, Obert L et al. Human and rodent bone marrow mesenchymal stem cells that express primitive stem cell
22
23
24
253
markers can be directly enriched by using the CD49a molecule. Cell Tissue Res 2007;327:47183. Tokalov SV, Gruner S, Schindler S et al. Age-related changes in the frequency of mesenchymal stem cells in the bone marrow of rats. Stem Cells Dev 2007;16:43946. Dominici M, Le Blanc K, Mueller I et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:3157. Alhadlaq A, Mao JJ. Mesenchymal stem cells: isolation and therapeutics. Stem Cells Dev 2004;13:43648.
Immunophenotype characterization of rat mesenchymal stromal cells MT Harting1,2, F Jimenez1,2, S Pati3,4, J Baumgartner1 and CS Cox1,2 1
Department of Pediatric Surgery and 2The Trauma Research Center, University of Texas Medical School at Houston, 3Department of Neurobiology and Anatomy, University of Texas Medical School at Houston, and The Vivian L. Smith Center for Neurologic Research, and 4 Department of Physical Medicine and Rehabilitation, Baylor College of Medicine, Houston, Texas, USA
Background Mesenchymal stromal cells (MSC) have shown diverse therapeutic potential. While characterization of human and mouse MSC has seen significant advances, rat bone marrow-derived MSC (rBM-MSC) remain under-characterized. We detail the isolation, expansion, differentiation, and detailed immunocharacterization of rBM-MSC. Methods Rat MSC were isolated and expanded in multipotent adult progenitor cell (MAPC) media, and cell-surface marker expression through 10 passages was used to characterize the population and multipotency was confirmed via differentation. Results By passage 3, rBM-MSC were found to be CD11b , CD45 , CD29 , CD49e , CD73 , CD90 , CD105 and Stro-1 ,
Introduction Mesenchymal stromal cells (MSC) have shown great promise as therapeutic agents in various fields of study [15]. However, varied isolation and expansion techniques, along with diverse methods of cell characterization, have yielded ill-defined and incomparable cellular populations. While characterization of human and mouse MSC has seen significant advances in the last few years [610], characterization of rat bone marrow-derived MSC (rBMMSC) has lagged behind. Rats serve as critical models in numerous diseases where cellular therapy is being studied. As these results are translated to clinical trials, ensuring consistent cellular populations is vital. Therefore, we
without the use of cell sorting. Media selection was responsible for the isolation of a nearly homogeneous population of rBM-MSC. The rBM-MSC immunophenotype changed by passage 10, showing decreases in CD73, CD105 and Stro-1 expression. Discussion Detailed characterization of cell populations facilitates accurate and reproducible cell therapy investigation. Given the expanding body of research involving rBM-MSC, these results advance our ability to compare rBM-MSC populations. Keywords cell therapy, immunocharacterization, marker expression, mesenchymal stromal cell, rodent, stem cell.
sought to isolate, expand and characterize the immunophenotype of rBM-MSC. Since the early 1970s MSC have been defined by their ability to adhere to plastic and proliferate [11]. Nearly 40 years later, this property remains the key step in the isolation of MSC. More recently, MSC have been defined further by their ability to differentiate down multiple lineages (most commonly adipocytes, chondrocytes and osteocytes) [6,12], including reports of differentiation into neural [13,14] and hepatic [15], among other, cell types. Cells similar to MSC, known as multipotent adult progenitor cells (MAPC), have been reported to differentiate down all three germ cell lines [16]. Others have
Correspondence to: Charles S. Cox, Jr, MD, Department of Pediatric Surgery, University of Texas Medical School at Houston, 6431 Fannin St, MSB 5.254, Houston, TX 77030, USA. E-mail: [email protected] – 2008 ISCT
DOI: 10.1080/14653240801950000
244
MT Harting et al.
defined MSC based on cell surface marker expression or lack thereof [7,8]. Most of the defining properties, markers and actions that characterize rBM-MSC were originally identified while using human and mouse cell lines [6,10,16]. Many of the same characteristics have been shown to transcend species, however some may not. We detail our methods for isolating rBM-MSC. We do not require magnetic-activated cell sorting (MACS) or fluorescent-activated cell sorting (FACS) to isolate our population of rBM-MSC. We follow three cell lines through to passage 10, characterizing the following cell-surface markers over time via flow cytometry: CD45, CD11b, CD29, CD49e, CD73, CD90, CD105 and Stro-1. A near homogeneous population of rBM-MSC is seen by passage 3 (14 days). By passage 8 or 9 (c. 1 month), cells develop suboptimal growth patterns and altered cell-surface marker expression. The rBM-MSC were differentiated to adipocytes, chondrocytes and osteocytes, confirming their multipotency.
Methods Cell isolation from BM SpragueDawley rats (200225 g) were purchased from Harlan Sprague Dawley (Indianapolis, IN, USA) for use in this study. The animals were housed on a 12-h light/dark cycle with ad libitum access to food and water. All protocols involving the use of animals were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (protocol HSC-AWC06038). Immediately after killing, the muscle was dissected from the femurs and tibiae, leaving isolated bone. The tips of the bones were shaved off with a Rongeur. Each bone was flushed with c. 10 ml of Medium 199 (Gibco, Carlsbad, CA, USA), leaving a cell suspension. After repetitive flushing (1015 times) through an 18-gauge needle, the cell suspension was filtered through a 40-mm nylon cell strainer (BD Falcon, Bedford, MA, USA). The cell suspension was then centrifuged for 6 min at 800 g. Cells were counted and plated in 6-well plates (Nunc, Rochester, NY, USA) coated with fibronectin (Sigma, St. Louis, MO, USA; 100 ng/mL in 1 PBS) at c. 106 cells/cm2 (c. 107 cells/well) in 2 mL media/well. Warm media (1 mL) were added after 48 h. After 72 h, all media were removed, the wells gently washed with 1 PBS, and refed with fresh, warm media. Media were replaced every 48 h for another 46 days. All
cells were then separated from the plastic (passage), using 0.05% trypsin EDTA (Cellgro, Manassas, VA, USA), every 4872 h, through to passage 10. See Figure 1 for an overview of cell isolation and expansion. Cells were maintained in a rat media mixture as described previously [17]. The rat media contains 60% low-glucose Dulbecco’s modified Eagle media (DMEM; Gibco), 40% MCDB-201 (Sigma), 1 insulin-transferrinselenium (ITS; Sigma), 1 linoleic acid bovine serum albumin (LA-BSA; Sigma), 10 9 m dexamethasone (Sigma), 10 4 m ascorbic acid 3-phosphate (Sigma), 100 U penicillin, 1000 U streptomycin (Gibco), 2% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 10 ng/ mL human platelet-derived growth factor (R&D Systems, Minneapolis, MN, USA), 10 ng/mL mouse epidermal growth factor (Sigma) and 1000 U/mL mouse leukemia inhibitory factor (Chemicon, Temecula, CA, USA). Cells isolated and expanded in the above media (MAPC media) were compared with cells isolated and expanded in basic media consisting of DMEM (low glucose; Gibco),supplemented with 10% FBS (HyClone) and penicillin/streptomycin (Gibco).
Flow cytometric characterization Flow cytometry analysis was performed using a BD LSR II (BD Biosciences, San Jose, CA, USA). Cells were collected from three SpragueDawley rats and flow cytometric analysis performed at passage zero (P 0 or BM) and P 1, 2, 3, 4, 5 and 10. For each passage, cells were detached with trypsin (as above) and labeled with a fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated primary antibody (Ab) or purified primary Ab, followed by a conjugated secondary Ab. The following cell-surface markers were characterized at each passage: CD11b, CD45, CD49e, CD73, CD90 (all from BD Biosciences), CD105 (Santa Cruz, Santa Cruz, CA, USA), CD29 (BioLegend, San Diego, CA, USA) and Stro-1 Bone Marrow Isolation
Remove non-adherent cells
Day 0
Day 3
1st Passage (P=1)
P=2
P=3
P=4
P=5
Day 7
Day 9
Day 11
Day 13
Day 15
P=10
Day 25
Flow Cytometric Analysis CD 11b CD 45 CD 29 CD 49e CD 73 CD 90 CD 105 Stro-1
Figure 1. Overview of cell isolation, expansion and immunophenotyping.
Immunophenotype characterization of rat MSC
(Zymed (now Invitrogen, Carlsbad, CA); Jackson ImmunoResearch, West Grove, PA, USA). Each Ab is specific to rat markers. Secondary Ab were purchased from BD Biosciences, Zymed or Jackson ImmunoResearch. Standard labeling protocols and the manufacturers’ suggestions were followed. Cells were suspended at 106 cells/mL and incubated with primary Ab (1:100, v/v) for 30 min at room temperature (RT). If not primarily conjugated, the cells were subsequently incubated with secondary Ab (1:100, v/v) for 30 min at RT. For the CD105 Ab, cells were fixed with 4% paraformaldehyde (PFA) for 30 min prior to incubation with primary Ab. The MSC population was identified based on forward/side scatter (FSC/SSC) properties. This population was gated to collect a maximum of 10 000 events.
Differentiation media/induction protocol/ staining Prior to differentiation, rBM-MSC were plated in rat media at 104 cells/cm2 in 12-well plates. The cells were allowed to adhere to the plastic and were near confluence after 2448 h. Undifferentiated rBM-MSC were stained using the identical protocols for Oil Red O (adipocytes), Alcian Blue (chondrocytes) and Alizarin Red S (osteocytes), as described below. These served as negative controls. For adipogenic differentiation, cells were plated in the presence of alpha MEM medium (Invitrogen, Carlsbad, CA, USA) containing 12.5% horse serum (Invitrogen), 12.5% FBS (HyClone), penicillin/streptomycin (50 U/ mL; Gibco), b-mercaptoethanol (10 4 m; Sigma) and hydrocortisone (10 4 m; Stem Cell Technologies, Vancouver, BC, Canada). The media were changed completely every 4872 h. The cells were allowed to differentiate for 1428 days. Characterization of adipocytes was performed by Oil Red O staining (Diagnostic Biosystems, Pleasanton, CA, USA) of the intracellular lipid-rich vacuoles. After fixation with 4% PFA for 30 min at RT, cells were washed and incubated in Oil Red O solution for 50 min at RT. After washing, cells were counterstained with Mayer’s hematoxylin for 5 min at RT. Cells were immediately imaged using inverted microscopy (Nikon, Melville, NY, USA). For chondrogenic differentiation, cells were cultured at 3 105 cells/mL in serum-free chemically defined medium consisting of DMEM (high-glucose; Invitrogen), 1 insulin-transferrin-selenium (Sigma), 1 LA-BSA
245
(Sigma), ascorbate 2-phosphate (50 mg/mL; Sigma), dexamethasone (100 nm; Sigma) and 10 ng/mL hTGF-b1 (R&D Systems). The media were changed completely every 4872 h. The cells were allowed to differentiate for 1428 days. Characterization of chondrocytes was performed by Alcian Blue staining (Richard Allen Scientific, Kalamazoo, MI, USA) of the proteoglycan-rich cartilage matrix. After fixation with 4% PFA for 30 min at RT, cells were washed and incubated in Alcian Blue solution for 30 min at RT. The staining solution was removed thoroughly via 34 distilled water washes. Cells were immediately imaged using inverted microscopy (Nikon). For osteocyte differentiation, cells were plated in the presence of osteogenic differentiation media (Lonza, Walkersville, MD, USA) that contained basal medium, dexamethasone, ascorbate, mesenchymal cell growth supplement, l-glutamine, penicillin/streptomycin and b-glycerophosphate. The media were prepared according to the manufacturer’s instructions. The media were changed completely every 4872 h. The cells were allowed to differentiate for 1428 days. Characterization of osteocytes was performed by Alizarin Red S indicator staining (Ricca Chemical Company, Arlington, TX, USA) for calcium formation. After fixation with 70% ice-cold ethanol for 1 h at RT, cells were washed and incubated in Alizarin Red S solution for 30 min at RT. The staining solution was removed thoroughly via 34 distilled water washes. Cells were immediately imaged using inverted microscopy (Nikon).
Analysis Flow cytometry data were collected using a BD LSR II and initial statistical analysis was performed using BD FACS Diva Software (BD Biosciences; Microsoft Excel (Microsoft, Redmord, WA, USA)). The data were further analyzed and graph overlays created using FlowJo software (version 7.2.1; Tree Star Inc., Ashland, OR, USA). Graphs of marker percentage versus passage number were generated using Microsoft Excel (Excel Professional 2003). All numbers are expressed as mean9SEM.
Results We initially identified rBM-MSC after 46 days of culture in media (Figure 2A). An additional few days (35 days, with media changes every 48 h) allowed the proliferating rBM-MSC to expand clonally without overcrowding. Rat MSC form a characteristic triangular or ‘fibroblastic’
246
MT Harting et al.
Figure 2. (A) Heterogeneous population of BM cells after 56 days in culture. MSC are distinguished by their characteristic spindle or fibroblastic shape when adherent to plastic. (B) A nearly homogeneous population of MSC (passage 3).
appearance when adherent to plastic (Figure 2B). Over subsequent passages the rBM-MSC maintained their morphological appearance. We found cell doubling times to range between 12 and 48 h, dependent upon passage number. After 910 passages a decreased growth rate and larger cell mass were noted. The newly isolated rat BM contained a mixture of cells identifiable by flow cytometry (Figure 3A, B). Cells found in the area where rBM-MSC are identified in future passages were largely positive for CD90, CD29, CD49e, CD73, CD105 and Stro-1; however, they were also largely positive for CD11b and CD45 (Figure 3B, C). Cells located in the mononuclear cell and lymphocyte areas were found to express CD90 (and the other positive markers above) while lacking CD11b (and CD45) expression (Figure 3B, C).
In the BM populations, we found some variability between different rats in the expression of the eight markers studied (Figure 4). In general, MSC have been defined as a population negative for CD11b and CD45. CD11b and CD45 were found on 34.790.74% and 75.69 8.58%, respectively, of newly isolated rat BM cells. Markers that are reported to be present on MSC were found on relatively small subsets of rat BM cells (Figure 4). CD73 and CD105 were found on less than 20% of cells, while other markers were noted on as many as 40% of the total cell population. Over the first three passages, rBM-MSC emerged from a heterogeneous population to a more homogeneous population (Figure 5AE). In the BM, cells positive for CD90 and negative for CD11b could be found in the mononuclear cell area (Figure 5A). After the first passage, few CD90 CD11b cells could be found (Figure 5B). By the second passage, the population expressing markers characteristic of MSC began to emerge (Figure 5C). An expanded population of CD90 CD11b cells could be seen by passages 3 and 4 (Figure 5D, E). Over the first three cell passages, CD11b and CD45 cells declined rapidly, each falling to less than 10% by passage 2, around 5% by passage 3 and less than 2% by passage 4. The percentage of cells expressing CD29, CD49e and CD90 rapidly increased to 90% by passage 2 and remained above 99% after passage 4. The other markers showed slightly increased variability. Markers CD73, CD105 and Stro-1 reached peak expression between passages 3 and 6. All markers showed somewhat decreased expression by passage 10. Cell marker expression over 10 passages is shown in Figure 4. Histogram overlays of each marker over the first three passages (until a near homogeneous population existed) are shown in Figure 6. Given previous reports of the inability to isolate a pure population of MSC, exclusive of hematopoietic stem cells or macrophages, in culture without cell sorting [8], we compared isolation and expansion in the MAPC media with isolation and expansion in a basic media consisting of DMEM, FBS and penicillin/streptomycin. We found that 2030% CD11b and CD45 cells remained after isolation and three passages in the basic media. Cells isolated and expanded through three passages in MAPC media, however, were found to be CD11b and CD45 (Figure 7).
Immunophenotype characterization of rat MSC
247
Figure 3. BM cell scatter plot (A) revealing the characteristic cellular populations (B). Although rMSC will eventually appear in the MSC area, a small fraction of cells in the monocyte or lymphocyte area are CD90 CD11b (C).
248
MT Harting et al.
CD 11b
60 40 20 0
0
2
4 6 Cell Passage number
8
10
CD 29 100 80 60 40 20 0
0
2
4 6 Cell Passage number
8
10
CD 73 100 80 60 40 20 0
0
2
4 6 Cell Passage number
8
10
CD 105 100 80 60 40 20 0
0
2
4 6 Cell Passage number
8
10
Percentage Positive for marker (%)
80
Percentage Positive for marker (%)
100
Percentage Positive for marker (%)
CD 45
Percentage Positive for marker (%)
Percentage Positive for marker (%)
Percentage Positive for marker (%)
Percentage Positive for marker (%)
Percentage Positive for marker (%)
D
100 80 60 40 20 0
0
2
4 6 Cell Passage number
8
10
8
10
8
10
8
10
CD 49e 100 80 60 40 20 0
0
2
4 6 Cell Passage number CD 90
100 80 60 40 20 0
0
2
4 6 Cell Passage number Stro-1
100 80 60 40 20 0
0
2
4 6 Cell Passage number
Figure 4. Cell-marker expression percentage through 10 passages for the eight surface markers analyzed. Cells from passage 3 display cell-surface marker expression characteristic of MSC. By passage 10, there is a decline in marker expression of CD73, CD105 and Stro-1.
Differentiation of the rBM-MSC to adipocytes, chondrocytes and osteocytes led to expected changes in cell morphology and staining characteristics, confirming their multipotency. Staining of passage 3 rBM-MSC controls with Oil Red O, Alcian Blue and Alizarin Red S identified no typical characteristics of adipocytes, chondrocytes or osteocytes, respectively (Figure 8A, D, G). After being exposed to the differentiation media, the cells developed the staining characteristics expected for adipocytes (Figure 8B, C), chondrocytes (Figure 8E, F) and osteocytes (Figure 8H, I). We found that c. 6070% of cells stained positive with Oil Red O (adipocytes) and that the differentiated adipocytes remained adherent to plastic. The chondrocytes gathered into large spheres that stained deeply positive
with Alcian Blue, consistent with human/mouse differentiation. Among individual chondrocytes, c. 5060% of the cells stained positive with Alcian Blue. Osteogenic differentiation led to a rapid phenotypic change toward cuboidal morphology and some delamination from the culture surface. In areas where cells remained adherent, the entire differentiated cell mass stained bright orange after staining with Alizarin Red S.
Discussion We have isolated and characterized a population of rBMMSC. We found that a nearly pure population of rBMMSC was achievable by passage 3, without the use of MACS or FACS. The cells continued to expand well and
Immunophenotype characterization of rat MSC
249
Figure 5. (A) Expansion of the rMSC population over the first four passages. The BM contains relatively few CD90 CD11b cells. (B) After passage 1, the number of CD90 CD11b cells remains relatively small. (C) By passage 2, the population of rMSC reaches 5075% of the total cellular population. The CD90 CD11b cellular population is 98% by passage 3 (D) and remains homogeneous in passage 4 (E).
250
MT Harting et al.
Figure 6. Overlays for all cell markers analyzed over three passages. The first column shows the FSC/SSC of the complete population, with a box around the gated cell population.
maintained a consistent phenotype, through passages 78. By passage 8 or 9, rBM-MSC had developed suboptimal growth patterns and immunophenotype changes. Given proper media conditions, the rBM-MSC were induced to differentiate down adipogenic, chondrogenic and osteogenic phenotypes, as evidenced by the development of specific staining characteristics.
The characterization of MSC has seen significant progress over the last decade. Human MSC isolated from the BM were initially and thoroughly characterized by Pittenger et al. in 1999 [6]. Human MSC isolated from other locations were subsequently characterized [18]. Murine MSC characterization followed shortly thereafter [10,19]. Rat MSC have been somewhat characterized [20
Figure 7. Overlay showing cell marker expression (passage 3) with MAPC media and basic media. After three passages in basic media, a significant cell population continues to express CD11b and CD45, while cells expanded in MAPC media are CD11b and CD45 negative.
Immunophenotype characterization of rat MSC
251
Figure 8. Rat MSC differentiation to adipocytes (AC), chondrocytes (DF) and osteocytes (GI). (A) Undifferentiated rMSC stained with Oil Red O at 10 (control); (B) adipocyte-differentiated rMSC stained with Oil Red O at 10 ; (C) adipocyte-differentiated rMSC stained with Oil Red O at 30 ; (D) undifferentiated rMSC stained with Alcian Blue at 4 (control); (E) chondrocyte-differentiated rMSC (spheroid) stained with Alcian Blue at 10 ; (F) chondrocyte-differentiated rMSC stained with Alcian Blue at 20 ; (G) undifferentiated rMSC stained with Alizarin Red S indicator at 4 (control); (H) osteocyte-differentiated rMSC stained with Alizarin Red S indicator at 4 ; and (I) osteocytedifferentiated rMSC stained with Alizarin Red S indicator at 10 .
22], although many seem to assume that they share similar characteristics to murine MSC. Through rigorous work by many groups, human/murine MSC have been described as generally positive or negative for the following markers (not species specific): CD11b , CD14 , CD34 , CD45 , CD13 , CD29, CD44, CD49e , CD71 , CD73, CD90 , CD105 , CD106 , CD120a , CD124 and Stro-1 . Given the limited availability of commercial Ab specific to the rat, we identified available Ab for these markers specific to rat cells. A complete understanding of the immunophenotype of species-specific cell populations is a key part of cellular
characterization. In 2006, Dominici et al. [23] outlined minimal criteria for defining human MSC. The criteria were: (1) adherence to plastic in standard culture conditions; (2) expression of CD105, CD73 and CD90 combined with a lack of expression of CD45, CD34 and CD14 or CD11b and CD79 or CD19 and HLA-DR surface molecules; and (3) differentiation into osteoblasts, adipocytes and chondroblasts in vitro. Cell characterization by surface molecule expression is rapid, highly specific and requires a small cell number. Immunophenotyping is likely to become the standard for cell characterization and comparison of cellular populations.
252
MT Harting et al.
Many reports of MSC isolation and characterization discuss the importance of cell sorting in order to develop a pure population of cells [7,8,12,16,24]. They propose the use of MACS or FACS in order to eliminate other hematopoetic or non-stem cells. They feel that without such sorting a heterogeneous population of cells, including hematopoetic stem cells and macrophages, will remain. Although non-MSC resilience is seen with rBM-MSC isolation, it has been particularly problematic when isolating mouse MSC. Schrepfer et al. [8] have shown that, in their cultures, macrophages are not effectively eliminated until after passage 10. Our data indicate that sorting may not be necessary to develop a homogeneous (95%) population of rBM-MSC. We show that MAPC media is probably responsible for this phenomenon. We found that 95% of the cells were CD11b , CD45 , CD29, CD49e, CD73, CD90, CD105 and Stro-1 by the third passage. Such a pattern of cell-surface marker expression is congruent with the surface markers previously characterized for human and mouse MSC. Given our findings, we make the following recommendations for rMSC isolation and expansion. (1) Rat BMMSC in passages 38 show the most uniform immunophenotypic characteristics (earlier passages show a mixture of phenotypic surface molecules and later passages experience phenotypic changes). (2) MACS and/or FACS may be unnecessary to achieve a nearly homogeneous population of rBM-MSC. Isolation and expansion in MAPC media leads to a more homogeneous population over few passages compared with the commonly used basic media. (3) Characterization of high-passage ( 810 passages) rBM-MSC by CD90 , CD29, CD49e , CD45 and/or CD11b alone may not accurately reflect the immunophenotype. (4) Rat BM-MSC were found to be CD11b , CD45 , CD29 , CD49e , CD73, CD90, CD105 and Stro-1 by passage 3; immunophenotypic characterization may become the standard for identifying and comparing rBM-MSC, given the high specificity, low cell number requirement and speed.
Acknowledgements We acknowledge The University of Minnesota Stem Cell Institute (Minneapolis, MN, USA), specifically Yuehua Jiang and Catherine Verfaillie (now in Belgium), for the generous opportunity to train in their NIH sponsored
program. Supported by NIH grant T32 GM008792-06 (M. T. Harting).
References 1 Oreffo RO, Cooper C, Mason C, Clements M. Mesenchymal stem cells: lineage, plasticity, and skeletal therapeutic potential. Stem Cell Rev 2005;1:16978. 2 Bussolati B, Camussi G. Adult stem cells and renal repair. J Nephrol 2006;19:7069. 3 Khakoo AY, Pati S, Anderson SA et al. Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi’s sarcoma. J Exp Med 2006;203:123547. 4 Leo AJ, Grande DA. Mesenchymal stem cells in tissue engineering. Cells Tissues Organs 2006;183:11222. 5 Pittenger MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res 2004;95:920. 6 Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:1437. 7 Gang EJ, Bosnakovski D, Figueiredo CA et al. Ssea-4 identifies mesenchymal stem cells from bone marrow. Blood 2007;109: 174351. 8 Schrepfer S, Deuse T, Lange C et al. Simplified protocol to isolate, purify, and culture expand mesenchymal stem cells. Stem Cells Dev 2007;16:1057. 9 Martinez C, Hofmann TJ, Marino R et al. Human bone marrow mesenchymal stromal cells express the neural ganglioside gd2: a novel surface marker for the identification of MSCs. Blood 2007;109:42458. 10 Tropel P, Noel D, Platet N et al. Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow. Exp Cell Res 2004;295:395406. 11 Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guineapig bone marrow and spleen cells. Cell Tissue Kinet 1970;3:393 403. 12 Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:714. 13 Brazelton TR, Rossi FM, Keshet GI, Blau HM. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000;290:17759. 14 Mezey E, Chandross KJ, Harta G et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:177982. 15 Petersen BE, Bowen WC, Patrene KD et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:116870. 16 Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:419. 17 Breyer A, Estharabadi N, Oki M et al. Multipotent adult progenitor cell isolation and culture procedures. Exp Hematol 2006;34:1596601.
Immunophenotype characterization of rat MSC
18 Mitchell JB, McIntosh K, Zvonic S et al. Immunophenotype of human adipose-derived cells: temporal changes in stromalassociated and stem cell-associated markers. Stem Cells 2006;24: 37685. 19 Meirelles Lda S, Nardi NB. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol 2003;123:70211. 20 Zangi L, Rivkin R, Kassis I et al. High-yield isolation, expansion, and differentiation of rat bone marrow-derived mesenchymal stem cells with fibrin microbeads. Tissue Eng 2006;12:234354. 21 Gindraux F, Selmani Z, Obert L et al. Human and rodent bone marrow mesenchymal stem cells that express primitive stem cell
22
23
24
253
markers can be directly enriched by using the CD49a molecule. Cell Tissue Res 2007;327:47183. Tokalov SV, Gruner S, Schindler S et al. Age-related changes in the frequency of mesenchymal stem cells in the bone marrow of rats. Stem Cells Dev 2007;16:43946. Dominici M, Le Blanc K, Mueller I et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:3157. Alhadlaq A, Mao JJ. Mesenchymal stem cells: isolation and therapeutics. Stem Cells Dev 2004;13:43648.