|Year : 2018 | Volume
| Issue : 2 | Page : 48-54
Which stem cells to choose for regenerative medicine application: Bone marrow and adipose tissue stromal stem cells – Similarities and differences
Nehad M Alajez1, Dalia Al-Ali2, Radhakrishnan Vishnubalaji1, Muthurangan Manikandan1, Musaad Alfayez1, Moustapha Kassem3, Abdullah Aldahmash4
1 Department of Anatomy, Stem Cell Unit, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia
2 Department of Anatomy, Stem Cell Unit, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia; Department of Endocrinology, Molecular Endocrinology Unit, University Hospital of Odense, University of Southern Denmark, Odense, Denmark
3 Department of Anatomy, Stem Cell Unit, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia; Department of Endocrinology, Molecular Endocrinology Unit, University Hospital of Odense, University of Southern Denmark, Odense; Department of Cellular and Molecular Medicine, Danish Stem Cell Center (DanStem), University of Copenhagen, Copenhagen, Denmark
4 Department of Anatomy, Stem Cell Unit, College of Medicine, King Saud University; Prince Naif Health Research Center, King Saud University, Riyadh, Kingdom of Saudi Arabia
|Date of Web Publication||6-Jun-2018|
Nehad M Alajez
Department of Anatomy, Stem Cell Unit, College of Medicine, King Saud University, Riyadh 11461
Kingdom of Saudi Arabia
Source of Support: None, Conflict of Interest: None
Background: Clinical use of stromal stem cells in regenerative medicine is increasingly recognized as a promising treatment modality for age-related degenerative diseases based on the promising initial results of clinical trials. However, the magnitude of positive effects observed in these trials has been variable which can be explained by the lack of standardization of the stem cell products “cell product.” Bone marrow-derived stromal (also known mesenchymal) stem cells (BM-hMSC) and adipose tissue-hMSC (AD-hMSC) have been used interchangeably in clinical trials employing stromal stem cells as they were thought to be functionally identical. Methods: In the present study, we performed an extensive side-by-side comparison of BM-hMSC and AD-hMSC for their CD marker expression using FACS analysis, molecular phenotype using global mRNA gene expression analysis, and functional studies for their in vitro differentiation capacity to osteoblasts and adipocytes. Results: We observed both stromal cell populations were CD44+ CD13+ CD90+ CD29+ CD105+ CD14− HLDR−. We also observed that they express common genetic signature consisting of 13,667 genes with enrichment in a number of pathways relevant to stem cell biology, for example, focal adhesion, insulin signaling, and mitogen-activated protein kinase signaling. On the other hand, we observed significant differences in their molecular phenotype with 3282 and 1409 genes differentially expression in BM-hMSC and AD-hMSC, respectively. Further analysis revealed higher expression of genes associated with osteoblast differentiation in BM-hMSC and those of adipocyte differentiation in AD-hMSC which correlated with their differential capacity for osteoblast versus adipocyte differentiation, respectively. Conclusion: Our data suggest that the clinical use of MSC in therapy depend on MSC site of origin, and thus, BM-hMSC are better suited for clinical trials aiming at enhancing bone regeneration. We suggest that molecular phenotype of stem cells is relevant approach for stem cell screening before their clinical transplantation.
Keywords: Adipose tissue, bone marrow, mesenchymal stromal cells, osteogenesis, pathways
|How to cite this article:|
Alajez NM, Al-Ali D, Vishnubalaji R, Manikandan M, Alfayez M, Kassem M, Aldahmash A. Which stem cells to choose for regenerative medicine application: Bone marrow and adipose tissue stromal stem cells – Similarities and differences. J Nat Sci Med 2018;1:48-54
|How to cite this URL:|
Alajez NM, Al-Ali D, Vishnubalaji R, Manikandan M, Alfayez M, Kassem M, Aldahmash A. Which stem cells to choose for regenerative medicine application: Bone marrow and adipose tissue stromal stem cells – Similarities and differences. J Nat Sci Med [serial online] 2018 [cited 2019 Jun 25];1:48-54. Available from: http://www.jnsmonline.org/text.asp?2018/1/2/48/233813
| Introduction|| |
Human stromal stem cells (commonly known as mesenchymal stem cells) (hMSCs) are adult multipotent stem cells that have the ability to differentiate into multiple mesodermal lineage cells, such as adipocytes, osteoblasts, and chondrocytes., MSCs are being introduced into a number of clinical trials for tissue repair, for example, bone and cartilage defects, and for the enhancement of tissue regeneration, for example, heart following myocardial infarction, brain following stroke, or immune modulation for example, graft-versus-host disease (GvHD). The standard site for obtaining human stromal cells is bone marrow (BM-hMSC) where the cells are located on the abluminal surface of blood vessels. However, obtaining sufficient samples to derived sufficient number of cells required for clinical studies is a major limitation for wide spread use of BM-hMSC. Over last several years, MSC-like populations have been obtained from a wide range of tissues, for example, adipose tissue, skin, umbilical cord blood, and placenta. Among all these tissues, adipose tissue is an attractive choice to obtain cells needed for clinical studies due to the ease of obtaining samples, during operative procedure, for example, liposuction. Human adipose-derived stromal stem cells (AD-hMSC) have been reported to exhibit a similar phenotype to that of BM-hMSC and have been suggested as an alternative source for obtaining MSC for clinical trials. However, a detailed analysis of the similarities and differences of these different cell populations at the molecular level has not clearly been defined.
The aim of the present study was to compare stromal cell populations obtained from human bone marrow and from human adipose tissue in terms of their phenotype, molecular profile, and their differentiation potential into osteoblasts and adipocytes.
| Materials and Methods|| |
Bone marrow-derived stromal (mesenchymal) stem cells were purchased from thermo fisher scientific (Thermo Fisher Scientific Life Sciences (Waltham, MA, USA). Adipose-derived mesenchymal stromal cells were isolated as described before.
The use of human specimens in the current study was approved by the Institutional Review Board at King Saud University College of Medicine (10-2815-IRB).
Cells were cultured in a basal culture medium of Dulbecco's Modified Eagle's medium (DMEM), supplemented with 4500 mg/L D-glucose, 4 mM L-glutamine, 110 mg/L 10% sodium pyruvate, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% nonessential amino acids. All reagents were purchased from Thermo Fisher Scientific Life Sciences (Waltham, MA, USA, http://www.thermofisher.com). Cells were incubated in 5% CO2 incubators at 37°C and 95% humidity.
Phenotype analysis was performed as previously described. In brief, trypsinized cells were washed twice in phosphate-buffered saline (PBS) supplemented with 0.5% FBS and resuspended to a concentration of about 1 × 105 cells/antibody test. For direct immunofluorescence, 10 μl FITC-conjugated mouse anti-human CD34, CD90, CD45, CD13, CD3, PE-conjugated mouse anti-human CD146, CD73, CD29, HLA-DR, and APC-conjugated mouse anti-human CD105, CD14, and CD44 antibodies (BD Biosciences, USA) were used. Nonspecific signal was analyzed by using a FITC/PE/APC-conjugated mouse IgG1 isotype antibodies, respectively. Cells were analyzed using BD FACSCalibur flow cytometer (BD Biosciences) and events were gated in a dot plot of forward versus side scatter signals on linear scale. At least, 10,000 gated events were acquired on a log fluorescence scale and data were analyzed using Kaluza Software Version 1.2 (Beckman Coulter, Indianapolis, IN).
Gene expression microarray
RNA isolation and gene expression analyses were carried out as described in our previously published manuscripts. In brief, RNA was isolated using the total tissue RNA purification kit from Norgen Biotek Corp., (Thorold, ON, Canada) and was quantified using NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). Total RNA was labeled and then hybridized to the Agilent Human SurePrint G3 Human GE 8 × 60 k microarray chip (Agilent Technologies, Santa Clara, CA, USA). All microarray experiments were conducted at the Microarray Core Facility (Stem Cell Unit, Department of Anatomy, King Saud University College of Medicine). Data were subsequently normalized and analyzed using GeneSpring 13.0 software (Agilent Technologies, Santa Clara, CA, USA). Pathway analyses were conducted using the Single Experiment Pathway analysis feature in GeneSpring 13.0 (Agilent Technologies).
The adipogenic induction medium (AIM) consisted of DMEM supplemented with 10% FBS, 10% horse serum (Sigma-Aldrich, St. Louis, MO, USA, http://www.sigmaaldrich.com), 1% penicillin/streptomycin, 100 nM dexamethasone, 0.45 mM isobutyl methyl xanthine (Sigma-Aldrich), 3 mg/mL insulin (Sigma-Aldrich), and 1 mM rosiglitazone (BRL49653). The AIM was replaced every 3 days. Cells were assessed for adipogenic differentiation on day 7.
Oil Red O and Nile Red staining
Adipogenic differentiation was determined by qualitative Oil Red O staining for lipid-filled mature adipocytes. Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, then incubated with freshly made and filtered (0.45 mM) Oil Red O staining solution (0.05 g in 60% isopropanol; Sigma-Aldrich) for 1 h at room temperature. Nile red fluorescence staining and quantification of adipogenesis was performed using a stock solution of Nile red (1 mg/mL) in DMSO that was stored at − 20°C and protected from light. Staining was performed on unfixed cells. Cultured differentiated cells were grown in polystyrene flat-bottom 96-well tissue culture-treated black microplates (Corning Inc., Corning, NY, USA, http://www.corning.com) and washed once with PBS. The dye was then added directly to the cells at a final concentration of 5 μg/mL in PBS, and the preparation was incubated for 10 min at room temperature, then washed twice with PBS. The fluorescent signal was measured using a SpectraMax/M5 fluorescence spectrophotometer plate reader (Molecular Devices Co., Sunnyvale, CA, USA, https://www.moleculardevices.com) using the bottom well scan mode, during which nine readings were taken per well using excitation (485 nm) and emission (572 nm) spectra. Oil Red and Nile red fluorescence was imaged using an EVOS Cell Imaging System (Thermo Fisher Scientific Life Sciences).
BM-hMSC were cultured as noted in the previous section and exposed to osteogenic induction medium (DMEM containing 10% FBS, 1% penicillin-streptomycin, 50 mg/mL L-ascorbic acid [Wako Chemicals GmbH, Neuss, Germany, http://www.wakochemicals.de/], 10 mM α-glycerophosphate [Sigma-Aldrich], 10 nM calcitriol [1a, 25-dihydroxy vitamin D3; Sigma-Aldrich], and 100 nM dexamethasone [Sigma-Aldrich]).
Alkaline phosphatase staining and activity quantification
We used a BioVision alkaline phosphatase (ALP) activity colorimetric assay kit (BioVision, Inc., Milpitas, CA, USA, http://www.biovision.com/) with some modifications. Cells were cultured in 96 well plates under normal or osteogenic induction conditions. On day 10, wells were rinsed once with PBS and fixed using 3.7% formaldehyde in 90% ethanol for 30 s at room temperature. The fixative was removed, and 50 μL of p-nitrophenyl phosphate solution was added to each well. The plates were incubated for 20–30 min in the dark at room temperature until a clear yellow color was developed. The reaction was subsequently stopped by adding 20 μL of stop solution. Optical density was then measured at 405 nm using a SpectraMax/M5 fluorescence spectrophotometer plate reader. For ALP staining, the cells were washed in PBS, fixed in acetone/citrate buffer, and incubated with ALP substrate solution (naphthol AS-TR phosphate 0.1 M Tris buffer, pH 9.0) for 1 h at room temperature. Images were taken using an EVOS Cell Imaging System (Thermo Fisher Scientific Life Sciences).
Statistical analyses and graphing were performed using Microsoft excel 2010 and GraphPad Prism 6.0 software (GraphPad, San Diego, CA, USA). P values were calculated using a two-tailed t-test.
| Results|| |
Phenotypic characterization of bone marrow-derived human stromal stem cell and adipose-derived human stromal stem cell
A panel of surface markers was utilized to immunophenotype BM-MSC versus AD-MSC using FACS analysis [Figure 1]a and [Figure 1]b. Both cell types were negative for the endothelial and hematopoietic lineage markers (CD34, CD45, CD14, CD31, and HLA-DR), whereas they were positive for the stromal cell-associated markers (CD13, CD29, CD44, CD73, CD90, and CD105). This CD markers' panel indicated that they were of nonhematopoietic or endothelial origin and expressed general stromal cells markers.
|Figure 1: Flow cytometry analysis shows the phenotypic resemblance of BM-MSC and AD-MSC. Primary BM-MSC (a) and AD-MSC (b) were collected and were stained for the indicated surface markers and were analyzed by flow cytometry. The percentage of positive population is indicated on each plot. BM-MSC: Bone marrow-derived human stromal stem cell, AD-MSC: Adipose-derived human stromal stem cell|
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Molecular profiling of bone marrow-derived human stromal stem cell and adipose-derived human stromal stem cell revealed common gene signature
Phenotypic data revealed similarities between BM-hMSC and AD-hMSC based on general surface markers; however, it is not clear whether the two cell populations exhibit similar molecular phenotype, i.e., molecular signature. Gene expression profiling of BM-hMSC versus AD-hMSC revealed clear separation of the two cell types [Figure 2]a. We observed similarities in gene expression between the two cell populations where a common signature consisting of 13,667 genes was expressed by the two cell populations [Figure 2]b. Pathway analysis of the common gene signature revealed enrichment in several pathways related to MSC biology such as focal adhesion, mitogen-activated protein kinase (MAPK), transforming growth factor beta (TGFb), and adipogenesis pathway. List of the top enriched pathways is illustrated in [Figure 2]c. Illustration of the FAK pathway with matched entities from the microarray data is shown in [Figure 3].
|Figure 2: Microarray gene expression profiling of BM-MSC versus AD-MSC. (a) Heat map analysis and unsupervised hierarchical clustering were performed on expressed genes in BM-MSC versus AD-MSC. Each column represents one replica and each row represents a transcript. Expression level of each gene in a single sample is depicted according to the color scale. (b) Venn diagram illustrating the overlap between genes expressed in BM-MSC and AD-MSC. (c) Illustration of the top twenty enriched pathways performed on genes commonly expressed by BM-MSC and AD-MSC. BM-MSC: Bone marrow-derived human stromal stem cell, AD-MSC: Adipose-derived human stromal stem cell|
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|Figure 3: Illustration of the FAK pathway. Illustration of the FAK pathway enriched in the commonly expressed genes in BM-MSC and AD-MSC. Color scale indicates the expression level. Matched entities from the microarray data are highlighted. FAK: Focal adhesion kinase, BM-MSC: Bone marrow-derived human stromal stem cell, AD-MSC: Adipose-derived human stromal stem cell|
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Bone marrow-derived human stromal stem cell is enriched in osteogenic genes while adipose-derived human stromal stem cell is more enriched in adipogenic genes
Although our data revealed high degree of similarities in gene expression profile of BM-hMSC and AD-hMSC, there existed significant differences [Figure 2]b where BM-hMSC differentially expressed 3282 genes whereas AD-MSC differentially expressed 1409 genes. Interestingly, when we compared the expression levels of a panel of osteogenic markers (BGLAP, DLX5, IGF2, TGFb1, TGFb2, and TGFBR2) as well as a panel of adipogenic markers (AdipoQ, CEBPA, CEBPB, FABP4, LPL, and PPARg) in both cell types, we observed higher expression of the osteogenic gene markers in BM-hMSC [Figure 4]a while the expression of adipogenic gene markers was higher in AD-hMSC [Figure 4]b.
|Figure 4: Expression of osteogenic and adipogenic gene markers in BM-MSC and AD-MSC. (a) Expression of a panel of osteogenic markers (BGLAP, DLX5, IGF2, TGFb1, TGFb2, and TGFBR2) in BM-MSC and AD-MSC based on microarray data. (b) Expression of a panel of adipogenic markers (AdipoQ, CEBPA, CEBPB, FABP4, LPL, and PPARg) in BM-MSC and AD-MSC based on microarray data. BM-MSC: Bone marrow-derived human stromal stem cell, AD-MSC: Adipose-derived human stromal stem cell|
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Bone marrow-derived human stromal stem cell exhibits higher osteogenic while adipose-derived human stromal stem cell exhibits higher adipogenic differentiation potential
Based on gene expression data, we determined differences in the differentiation potential to osteoblasts and adipocytes between BM-hMSC and AD-hMSCs. Both cell populations were induced into osteoblastic cells, and on day 10, cells were stained for ALP activity. As shown in [Figure 5]a, higher levels of the osteoblastic marker ALP were observed in BM-hMSC compared to AD-hMSC. Concordantly, ALP enzymatic activity quantification revealed higher ALP activity in BM-hMSC compared to AD-hMSC [Figure 5]b. The adipogenic differentiation capacity of both cells types was also investigated. Enhanced adipogenic differentiation of AD-hMSC as compared to BM-hMSC based on quantification of mature adipocytes stained positive for Nile red was observed [Figure 5]c and [Figure 5]d.
|Figure 5: Differential osteoblastic and adipocytic differentiation of BM-MSC and AD-MSC. (a) Representative ALP staining on day 10 induction of BM-MSC (left panel) or AD-MSC (right panel). (b) Quantification of ALP activity in BM-MSC versus AD-MSC induced into osteoblast for 10 days. Data are presented as mean ± standard error of the mean; n = 8 from two independent experiments. ***P < 0.0005. (c) Representative Nile Red staining of lipid filled adipocytes on day 7 induction of BM-MSC (left panel) or AD-MSC (right panel). Images were captured using EVOS FL Auto system (Thermo) using ×10 objective. (d) Nile red quantification on day 7 after adipocytic induction of BM-MSC and AD-MSC. Data are presented as mean ± standard error of the mean; n = 6 from two independent experiments. ***P < 0.0005. BM-MSC: Bone marrow-derived human stromal stem cell, AD-MSC: Adipose-derived human stromal stem cell, ALP: Alkaline phosphatase|
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| Discussion|| |
There is an increasing interest in using stem cells in treatment of degenerative and age-related diseases, for example, Parkinson's disease, liver failure, leukemia, diabetes, osteoarthritis, and osteoporosis, for which there is no curative therapy. Furthermore, the need for novel approaches based on stem cell transplantation to enhance skeletal tissue regeneration and repair in cases of nonhealing fractures and bone defects is needed. The choice of functionally relevant cell type is important for the successful use of cells in therapy. Currently, bone marrow MSC and adipose tissue MSC were used interchangeably with the assumption that these cells are functionally similar. In the current manuscript, we demonstrate that these two cell population exhibit significant differences in their molecular phenotype and their functional differentiation capacity which is relevant to their clinical use.
The cellular phenotype of both BM-hMSC and AD-hMSC based on CD marker expression was similar suggesting that this cellular phenotype is characteristics of the stromal cell populations irrespective of their tissue of origin. Our data thus corroborate previous studies which exhibit similar panel of CD-surface markers known to be present in stromal cell populations. Similar to the presence of a common CD markers signature, both BM-hMSC and AD-hMSC shared a large number of genes and enrichment in a number of genetic pathways that are required for stem cell function, for example, focal adhesion signaling, insulin signaling, and MAPK signaling. Their functions include self-renewal capacity of stem cells (MAPK), providing sufficient energy (insulin signaling) and cellular identity in their niche (Focal adhesion).
We observed also significant differences between BM-hMSC and AD-hMSC in terms of significantly enriched gene groups. Interestingly, BM-hMSC has higher expression of genes relevant to osteoblast differentiation while AD-hMSC has higher expression of genes relevant to adipocyte differentiation. Interestingly, genes that were unique to BM-hMSC were more enriched in cell cycle regulation, while that were unique to AD-hMSC were more enriched in immune modulation (data not shown). MSCs were first described as nonhematopoietic, plastic adherent, multipotent, mesodermal germ layer-derived cells by Friedenstein et al. Interestingly, Friedenstein et al. termed bone marrow MSC as “committed osteoprogenitor” cells while MSC derived from other tissues as “inducible osteoprogenitor” based on their in vivo transplantation studies and the need of the “Inducible osteoprogenitor” cells for osteoblastic induction using growth factors, for example, bone morphogenetic proteins to reveal their osteogenic differentiation capacity. Our data provide molecular explanation for this phenomenon and demonstrate that AD-hMSC are poor are osteoblast differentiation compared to BM-hMSC.
We demonstrated that employing global gene expression of cultured cells, i.e., determining their molecular signature is predictive for their functional capacity. The molecular signature of BM-hMSC suggested commitment to osteoblastic differentiation and AD-hMSC suggested commitment to adipocytic differentiation which was confirmed in subsequent function studies. Our data may thus encourage using global gene expression analysis as an approach to determine the functional capacity of the cells before their use in clinical trials.
| Conclusion|| |
Our results have a clinical relevance as it demonstrates that “not all stem cells are equal” and thus proper choice of stem cells based on their expected functions following in vivo transplantation is needed. While the initial results of MSC used in clinical trials are promising, the magnitude of positive effects has been variable and likely caused by differences in the MSC populations employed as well as the lack of standardization of the MSC “cell product.” We suggest that in MSC cell-based therapy, it is important to employ well-characterized cell populations based on their molecular and functional phenotype that are aligned with the aim of their clinical use.
We would like to thank the Deanship of Scientific Research at King Saud University (Research Group No. RG-1438-033).
Financial support and sponsorship
This study was financially supported by Deanship of Scientific Research at King Saud University (Research Group No. RG-1438-033).
Conflicts of interest
There are no conflicts of interest.
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