Quantifying Mesenchymal Stem Cells in the Mononuclear Cell Fraction of Bone Marrow Samples Obtained for Cell Therapy

Quantifying Mesenchymal Stem Cells in the Mononuclear Cell Fraction of Bone Marrow Samples Obtained for Cell Therapy M. Alvarez-Viejo, Y. Menendez-Menendez, M.A. Blanco-Gelaz, A. Ferrero-Gutierrez, M.A. Fernandez-Rodriguez, J. Gala, and J. Otero-Hernandez ABSTRACT Aims. The use of bone marrow mononuclear cells (BMMNCs) as a source of mesenchymal stem cells (MSCs) for therapy has recently attracted the attention of researchers because BMMNCs can be easily obtained and do not require in vitro expansion before their use. This study was designed to quantify the MSC population in bone marrow (BM) samples obtained for cell therapy using flow cytometry to detect the CD271 antigen. Material and Methods. Autologous BM was obtained by posterior superior iliac crest aspiration under topical anesthesia. Mononuclear cells isolated from the BM aspirate on a Ficoll density gradient were used to treat patients with pressure ulcer (n ⫽ 13) bone nonunions (n ⫽ 3) or diabetic foot ulcers (n ⫽ 5). Results. Our flow cytometry data revealed a low percentage as well as a high variability among patients of CD271⫹CD45⫺ cells (range, 0.0017 to 0.0201%). All cultured MSC adhered to plastic dishes showing a capacity to differentiate into adipogenic and osteogenic lineages. Conclusions. Our findings suggested that the success of cell therapy was independent of the number of MSCs present in the BM aspirate used for autologous cell therapy. URING the past 10 years stem cell– based therapy has emerged as a promising tool for tissue engineering and repair procedures. Research efforts in regenerative medicine have mainly focused on the use of mesenchymal stem cells (MSCs), also known as marrow stromal cells,1 mesenchymal progenitor cells,2 or most recently, multipotent mesenchymal stromal cells.3 MSCs are defined as self-renewing, multipotent progenitor cells that are present in many adult tissues including bone marrow (BM),4 trabecular bone,5 and adipose tissue.6 MSCs can proliferate and differentiate into multiple mesodermal tissues such as bone, cartilage, muscle, tendon, and fat.7–9 Because of their potential for multilineage differentiation, MSCs are ideal candidates to replace damaged tissues.10 –12 Moreover, the MSC phenotype, which has been widely described to be MHC I⫹, MHC II⫺, CD40⫺, CD80⫺, and CD86⫺ is regarded to be nonimmunogenic. Therefore, their transplantation into an allogeneic host may not require immunosuppression.13 These characteristics make these cells promising candidates for new therapeutic strategies. Since their first observation in BM,14 MSCs have been routinely isolated based on their capacity to adhere to

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plastic plates. However, this isolation strategy risks hematopoietic cell contamination. Attempts have been reported to purify these cells directly from harvested BM.15 The in vivo phenotype of bone marrow mesenchymal stem cells (BMMSCs) has been difficult to establish because of the low frequency of MSCs in a BM aspirate (0.001% to 0.01%).4 To date, the exact phenotype of BMMSCs before culture has remained uncertain; recently the low-affinity nerve growth factor receptor (LNGFR; also known as (CD271) has been shown to be specifically expressed by these From the Transplant and Cell Therapy Unit (M.A.-V., Y.M.-M., A.F.-G., J.O.-H.), OIB-FICYT-HUCA (M.A.B.-G.), and the Haematology Unit (M.A.F.-R., J.G.), Hospital Universitario Central de Asturias, Oviedo, Spain. Supported by the Obra Social Cajastur and a project grant awarded by the FICYT PEST08-12. M. Alvarez-Viejo and Y. Menendez-Menendez, these authors have contributed equally in this work. Address reprint requests to Maria Alvarez-Viejo, Laboratorio de Trasplantes y Terapia Celular, Edificio Polivalente A, 1a planta, Hospital Universitario Central de Asturias, 33006 Oviedo, Asturias, Spain. E-mail: [email protected] © 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 45, 434 – 439 (2013)

QUANTIFYING MESENCHYMAL STEM CELLS

MSCs.16 Although CD271 was initially believed to be expressed by cells of the nervous system and play a key role in the development, survival, and differentiation of neural cells, it is now known to be a member of the low-affinity neurotrophin receptor and tumor necrosis factor receptor superfamily.17 Thus, CD271 has been proposed to be a versatile marker to selectively isolate and expand multipotent MSCs with both immunosuppressive and lymphohematopoietic engraftment-promoting properties.18,19 Moreover, according to some workers,20 non-expanded mesenchymal progenitors do not express the hematopoietic markers CD34, CD45, and glycophorin A. Transplantation of MSCs from BM is considered to be a safe procedure based on clinical trials.21 They have been deployed in almost all of the major organs of the body: heart, brain, lung, liver, and kidney.22 More recently, encouraging results have been achieved with autologous bone marrow mononuclear cells (BMMNCs) for treatment of severe peripheral arterial disease, nonacute ischemic stroke, and bone nonunion or avascular necrosis.23–25 Clinical studies have focused on the use of the entire BMMNC fraction, assuming that functional effects depend on a correct balance among multiple cell types and stem cell precursors.26 In addition, the cells and precursors of the BMMNC fraction produce large amounts of cytokines and trophic factors.24 BMMNCs are easily obtained and isolated in a short time just before transplantation, minimizing the risks of contamination. However, in most clinical studies, the transplanted progenitor cells have not been quantified before administration. We have obtained encouraging results using BMMNCs to treat patients with pressure ulcers,27 long bone nonunions, or ischemic vascular disease. The aim of this study was to quantify the proportion of CD271⫹ CD45⫺ cells among the mononuclear cell (MNC) fraction transplanted into each patient examining possible links to treatment outcomes, patient sex, or age.

435 suspension using a catheter into a created pouch seeking to revitalize the margins to close the wound. In bone nonunion cases, we implanted the cell suspension at the site of bone damage and fixation. To treat diabetic foot ulcers, we injected the cell suspension into an appropriate artery.

BMMNC Flow Cytometry Approximately 2.5 ⫻ 106 cells were separated from the extracted BM to identify the CD271⫹ population using flow cytometry (FACSAria analyzer and FACSDiva v6.1.3 software, Becton Dickinson, NJ, United States). The MNC suspension was washed twice in 1⫻ phosphate-buffered saline (PBS) (PAA Laboratories GmbH, Cölbe, Germany) supplemented with 3% fetal bovine serum (FBS, Gibco Invitrogen). Next, cells resuspended in a final volume of 100 ␮L were incubated for 20 minutes at 4°C with 20 ␮L FcR blocking reagent (eBioscience, Calif, United States) to avoid nonspecific antibody binding. Antibodies against human CD271-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) and human CD45-PE-Cy7 (eBioscience, California, United States) were then added followed by incubation for 30 minutes at 4°C. As isotype controls, we used IgG1-PE (Miltenyi Biotec) and IgG1-PE-Cy7 (eBioscience). Once labelled, the cells washed twice in 1⫻ PBS (PAA Laboratories) containing 3% FBS were resuspended in 0.5 mL 1⫻ PBS (PAA Laboratories). Before cytometry, cells were sieved through a 35-␮m nylon filter (Cell Strainer, Becton Dickinson). To obtain viable cell counts, we identified dead cells by labelling with 7-actinomycin D (BD Bioscience).

MSC Cultures Ficoll-separated BMMNCs were cultured by seeding 10 ⫻ 106 BM cells onto 25 cm2 polystyrene flasks (Cultek, Madrid, Spain) in 10 mL of DMEM supplemented with 10% FBS, 100 U/mL penicillin (Gibco Invitrogen), and 100 ␮g/mL streptomycin (Gibco Invitrogen). Cells were cultured in a controlled atmosphere (37°C, 5% CO2) for 48 hours before replacing the medium and removing unattached cells. Thereafter, the medium was replenished every 2 to 3 days. The confluent cells on days 10 to 14, were digested with trypsin (0.25% w/v; Gibco Invitrogen) before passage.

MSC Flow Cytometry MATERIALS AND METHODS Isolating BMMNCs BM aspirates obtained from 21 patients were used to treat pressure ulcers (n ⫽ 13) diabetic foot ulcers (n ⫽ 5), and long bone nonunions (n ⫽ 3). The therapeutic protocol was approved by our ethics committee in accordance with Spanish law. All patients signed a detailed informed consent form before any intervention. Consent was also obtained for publication of results from the study. Autologous BM was obtained by posterior superior iliac crest aspiration under topical anesthesia (Mepivacaine 2%, B. Braun, Melsungen, Germany) using a heparin-rinsed (Mayne heparin 25000 IU, Chicago, Ill, United States) trocar and syringe. MNCs were isolated from the BM aspirate on a Ficoll density gradient (Biocoll separating solution, 1.077 g/mL, Biochrom AG, Berlin, Germany) after centrifugation for 25 minutes at 400 g. After washing twice in RPMI medium (Mediatech, Va, United States), cells resuspended in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Invitrogen, Paisley, United Kingdom) were counted in a Neubauer hemocytometer. The cell infusion was always performed in the operating room. Patients with pressure ulcers underwent injection of the cell

The antibodies that were used against the surface marker panel were proposed by The Mesenchymal and Tissue Stem Cell Committee of the International Society for Celullar Therapy.28 The MSC phenotype was identified using antibodies against CD34PerCpCy5.5 (BD Bioscience), CD73-Pe (BD Pharmigen), CD90FITC (eBioscience), CD105-APC (eBioscience) and CD45-PeCy7 (eBioscience).

Differentiation of MSC Adipogenic differentiation was induced using NH AdipoDiff Medium (Miltenyi Biotec). In brief, MSCs resuspended at a final concentration of 5 ⫻ 104 cells/mL in NH AdipoDiff medium were seeded on 35-mm plates with fresh medium every third day. After 2 to 3 weeks, large vacuoles started to appear. Adipogenic differentiation was assessed on day 21, by Oil Red-O staining (SigmaAldrich, Madrid, Spain). Osteogenic differentiation was induced using NH OsteoDiff Medium (Miltenyi Biotec). MSCs were resuspended at a final concentration of 3 ⫻ 104 cells/mL in NH OsteoDiff medium and seeded onto 35-mm plates. The medium was replenished every third day. On day 10, differentiated cultures were stained with Sigma Fast Bcip/Nbt substrate (Sigma-Aldrich).

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Statistical Analysis

Clinical Results

Data are shown as mean values ⫾ standard errors. The significance level was set at a probability value of ⬍.05. After confirming the normal distribution of the data using the Kolmogorov-Smirnov test, mean values were compared using the Welch t test.

Our results for the use of autologous MNCs to treat pressure ulcers have been recently published.27 Using imaging procedures (nuclear magnetic resonance [NMR] or computed tomography [CT]), treatment success was close to 90%. Our data on treatment of long bone nonunions (unpublished) indicated a 100% success rate. Good preliminary results have also been observed by angiographic analysis of patients with diabetic foot ulcers, although the number of patients examined is still small. Considering the variability observed among patients, the number of transplanted MSCs seemed to be independent of the success of the therapy.

RESULTS Flow Cytometry Analysis of BMMNCs

Samples were isolated from autologous BM extracts obtained from each patient. The variables of gender, age, treated condition, and percentage of CD271⫹ CD45⫺ cells in the MNC fraction are shown for each patient in Table 1 The percentage of cells that were the appropriate phenotype ranged from 0.0017% to 0.0201% (Table 1). Low proportions and high variability of cells with the MSC phenotype were observed among the patients, as shown in Fig 1, which provides flow cytometry images for the two patients showing the highest (Fig 1A) and the lowest (Fig 1B) proportions of CD271⫹ CD45⫺ cells. Flow cytometry data refer to live cells (mean viability, 94.2% to 99.7%) determined using 7-actinomycin D. Because patient age varied widely (range, 30 to 78 years), numbers of MSCs were grouped by patients younger or older than 55 years; no significant difference was detected between the groups (P ⫽ .133, Table 2). There was no significant difference between men and women (P ⫽ .715, Table 2). MSC counts did not differ significantly according to the treated condition (P ⫽ .159, Table 2). Notwithstanding, patients with pressure ulcers showed a higher mean number of CD271⫹CD45⫺ than the others albeit this was not significant (Fig 2).

Characteristics of Cultured MSC

A sample of each patient’s Ficoll gradient-isolated MNCs was expanded in vitro to characterize the MSCs according to the criteria described by The Mesenchymal and Tissue Stem Cell Committee of the ISCT.28 Under standard culture conditions, all MSC cultures adhered to treated plastic maintaining their fibroblast-like morphology, as originally described by Friedenstein et al (1970).14 Passage 4 – 8 MSCs were assessed for their potential to differentiate into adipogenic and osteogenic lineages. All expanded MSCs were able to differentiate into these two lineages as revealed by labelling with specific stains. Adipogenesis was indicated by the deposition of neutral lipid vacuoles that stained with Oil Red-O; osteogenesis, by increased alkaline phosphatase-positive cells. The differentiation potential of MSCs obtained from each patient did not vary. Passages 3–5 MSCs immunolog-

Table 1. Patient Characteristics and Percentage CD271ⴙ CD45ⴚ Identified in Bone Marrow Aspirates Patient No.

Condition Treated

Gender

Age (y)

Total MSCs

% CD271⫹ CD45⫺

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Bone nonunion Pressure ulcer Pressure ulcer Pressure ulcer Pressure ulcer Bone nonunion Bone nonunion Pressure ulcer Pressure ulcer Pressure ulcer Pressure ulcer Pressure ulcer Pressure ulcer Pressure ulcer Diabetic foot ulcer Diabetic foot ulcer Diabetic foot ulcer Diabetic foor ulcer Pressure ulcer Pressure ulcer Diabtetic foot ulcer

Male Male Male Female Male Male Female Male Female Male Male Male Male Male Female Male Male Female Male Male Female

30 62 54 36 66 39 70 78 75 31 38 35 75 42 70 73 76 75 65 65 78

7392 50,544 15,300 9300 29,657 11,945 2160 2688 57,888 11,520 3000 112,700 16,284 7920 9555 4982 2406 2700 16,324 17,035 22,800

0.0022 0.012 0.0017 0.0025 0.016 0.011 0.0018 0.0032 0.020 0.0080 0.0025 0.012 0.0059 0.0060 0.0075 0.0048 0.0021 0.0027 0.0083 0.0084 0.0057

Abbreviation: MSC, mesenchymal stem cell.

QUANTIFYING MESENCHYMAL STEM CELLS

437

Fig 1. Flow cytometry analysis conducted on BMMNC samples from: (A) Patient 9, in whom the highest percentage of CD271⫹ CD45⫺ cells was observed, and (B) Patient 3, who showed the lowest percentage of CD271⫹ CD45⫺ cells.

ically characterized by cytometry, all were positive for the markers CD73, CD90, and CD105 and negative for the hematopoietic markers CD34 and CD45 (Fig 3). DISCUSSION

Despite the success of many procedures to treat damaged tissue, the mechanisms whereby the cell preparations exert their actions are still poorly understood. Prompted by the recent encouraging results to treat pressure ulcers in patients with spinal cord injury and long bone nonunions (unpublished data) using BMMNC at our center, we sought to characterize the CD271⫹ MSC population among BM samples obtained for these patients. Flow cytometry is a well-established tool for cell characterization studies.29 We identified the mesenchymal cell population in BMMNC samples using the CD271 antigen as a marker. Quirici et al reported that LNGFR antibodies are specific for isolating adult MSC from BM, suggesting that they can obtain multipotent cells.30 Although the phenotype of nonexpanded MSC is not clear, CD271 is a differentially expressed marker of BMMSC in vivo. Several

independent studies have indicated high-levels of CD271 expression among fresh BMMSC.16,30 Our flow cytometry results shown in Fig 1 reflected the highly variable proportion of CD271⫹ CD45⫺ cells detected among our patients. Figure 1A shows a distinct population of CD271⫹ CD45⫺ cells and Fig 1B, a much reduced number of these cells. In contrast, the BMMSC population showing the phenotype CD271⫹ CD45dim observed by Jones et al31 was described to be much less variable. The variability observed herein was consistent with the findings of other workers.4 Although we speculated a relationship between the number of transplanted CD271⫹ cells and healing responses a criterion that would serve to

Table 2. Summarized Patient Characteristics According to the Condition Treated, Gender, and Age Characteristic

N

CD271⫹CD45⫺ Cells*

P Value

Patients Male Female Condition treated Pressure ulcer Diabetic foot ulcer Bone nonunion Age ⬎56 years ⱕ55 years

21 15 6

14,889.08 ⫾ 3259.429 13,884.4 ⫾ 3210.925 17,400.50 ⫾ 8648.583

.715

13 5 3

19,133.12 ⫾ 4741.649 8488.60 ⫾ 3799.707 7165.67 ⫾ 2826.952

13 8

18,078.74 ⫾ 5075.785 9705.74 ⫾ 1307.412

.159

.133

*The values are given as the mean ⫾ typical error.

Fig 2. Mean ⫾ standard error of the number of mesenchymal stem cells detected in each patient according to the condition treated.

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Fig 3. Flow cytometry analysis of surface markers. Cells expressed CD73, CD90, and CD105 and lacked the expression of the hematopoietic markers CD34 and CD45. Panel (A) cytometry images corresponds to the isotype control, and panel B to the patient sample tested.

assess the quality of a BM sample for subsequent transplantation, we were unable to confirm this hypothesis. The observed high variation also seemed to be independent of sex, age, and disease. Stolzing et al observed MSC numbers obtained by marrow aspiration to diminish with age.32 They described an age-related decline in overall BMMSC “fitness” that could lead to problems when using autologous aged MSC for cell-based therapies.33 The discrepancy from our findings was likely attributable to the heterogeneity of our study population and the small number of patients. In addition to BM, MSCs have been identified in adipose tissue,6,34 liver, lungs, spleen,35,36 amniotic fluid,37 umbilical cord blood,38 umbilical cord,39,40 placenta,41,42 human endometrium,43 and dental pulp.44 However, the various sources and isolation methods have yielded inconsistent defining characteristics among investigators. To resolve this problem, The Mesenchymal and Tissue Stem Cell Committee of the ISCT proposed a set of standards to define human MSC for both laboratory-based scientific investigations and preclinical studies. The 3 criteria defining MSC are adherence to plastic in standard culture conditions, specific surface antigen expression, and multipotent differentiation potential.28 Our MSC populations that were identified in 21 BM aspirates showed in vitro adhesion to treated plastic and expression of CD73, CD90, and CD105 with lack of expression of the hematopoietic markers CD34 and CD45. Moreover, we also confirmed their capacity to differentiate into osteoblasts and adipocytes. In addition, despite the interpatient variation observed in the number of MSCs present in each BMMNC fraction, no variation was detected among patients in the in vitro characteristics of their MSCs. In conclusion, the variability of CD271⫹CD45⫺ cell percentages observed among patients could not be corre-

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