Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production
- Emily T. Camilleri1,
- Michael P. Gustafson2,
- Amel Dudakovic1,
- Scott M. Riester1,
- Catalina Galeano Garces1,
- Christopher R. Paradise1,
- Hideki Takai3,
- Marcel Karperien4, 5,
- Simon Cool6, 7,
- Hee-Jeong Im Sampen8, 9, 10, 11,
- A. Noelle Larson1,
- Wenchun Qu12,
- Jay Smith13, 14, 15,
- Allan B. Dietz2 and
- Andre J. van Wijnen1, 16Email author
© The Author(s). 2016
Received: 8 May 2016
Accepted: 20 July 2016
Published: 11 August 2016
Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL).
In this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors.
We characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells.
Our study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria.
Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007. Registered August 26, 2013.
MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540. Registered April 23, 2013.
Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027. Registered October 31, 2014.
Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145. Registered August 17, 2006.
A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283. Registered May 18, 2012.
Regenerative medicine endeavors to surpass traditional treatments through the use of biological products to restore damaged or diseased tissues that are otherwise beyond repair. Integral to many regenerative therapeutic strategies is the use of adult stem cells, and particularly the use of mesenchymal stromal cells (MSCs) for either their regenerative or immune-regulatory properties . Currently, over 246 clinical trials utilizing allogenic or autologous MSCs  are being performed worldwide to treat various diseases including osteoarthritis , atherosclerotic renal artery stenosis (ARAS) , multiple system atrophy (MSA) , graft versus host disease , and amyotrophic lateral sclerosis (ALS) . Despite the growing number of clinical trials, there are currently no US FDA-approved MSC-based products . Challenges for clinical translation of MSC-based therapies largely lie in the production and basic characterization of the MSC product .
Cell therapy using MSCs is largely limited by the cell harvesting and manufacturing of the MSC product. In recent years, cell therapy research has moved away from embryonic stem cells and induced pluripotent stem cells due to ethical and safety concerns over these cell types. Various alternative tissue sources of MSCs have been identified, including bone marrow, adipose tissue, umbilical cord, Wharton’s jelly, and gingival tissue. The majority of studies utilize MSCs derived from the bone marrow (BMSCs) owing to their potential to differentiate into various mesenchymal tissues, including bone, cartilage, and fat, as well as their immune-regulatory functions. However, bone marrow as a source of MSCs is limited by the invasive and painful aspiration procedure, and very low abundance of MSCs that typically account for 0.001–0.01 % of cells . Adipose tissue has been identified as an MSC-rich tissue, in which 1–10 % of the stromal fraction is MSCs [9, 10], which also undergo multi-lineage differentiation in vitro [9–13]. These attributes are advantageous and also permit autologous transplantation, which is particularly important for non-fatal diseases (e.g., wound healing, osteoarthritis, or aesthetic procedures). Although the anatomical location of harvesting may have some impact on the yield of adipose-derived MSCs (AMSCs) [14, 15], variability in processing, manufacturing, and delivery of MSC/AMSCs may have larger implications on cell therapy outcomes .
AMSCs preparations for cell therapy vary from minimal processing (isolation of the stromal vascular fraction) to ex vivo expansion of the processed lipoaspirate . The ex vivo expansion of AMSCs from the processed lipoaspirate is performed with either fetal bovine or calf serum (FBS or FCS), or under nonzoonotic conditions using human platelet lysate (hPL) [12, 17]. Previous studies have shown that culturing AMSCs in good manufacturing practices (GMP)-grade hPL provides a growth advantage, and the cellular yields were significantly greater for AMSCs grown in 5 % hPL compared to 10 % FBS or FCS [12, 17]. Tissue culture practices may also influence AMSC growth, where contact inhibition and/or cryopreservation may affect their function [18–20]. Finally, the therapeutic delivery of MSCs also varies among clinical trial protocols; MSCs are commonly cryopreserved, thawed, and administered, or allowed to recover in culture for up to 4 days prior to administration. It is currently not known how preparation procedures prior to administration may impact the function of MSCs following infusion or application.
Despite differences in isolation, production, and administration, characterization of an MSC-based product is largely limited to measuring the expression of a subset of classical cell surface markers, including CD90, CD73, CD105, and CD44, and absence of expression of CD45 or CD31 as defined by the International Society for Cellular Therapy (ISCT) and the International Federation of Adipose Therapeutics and Sciences (IFATS) [2, 11]. These markers only really serve to identify cells as MSCs so additional markers are needed to get information regarding potency and function of the cells, the differentiation potential, and how cultured cells change over time during manufacturing. To gain a better understanding of the MSC surface proteome, techniques including mass spectroscopy- and flow cytometry-based antibody screening assays have been used to characterize AMSC surface proteins and to determine the heterogeneity of MSC populations [21–26]. While these techniques are highly relevant for screening purposes, these studies have significant limitations in that they rarely utilize clinical-grade AMSCs or report whether the cells maintain homogeneity during manufacturing steps. As such, product characterization remains an unmet need for translational therapies using AMSCs. In this study, we utilized clinical-grade AMSCs grown in GMP- hPL, characterized the surface marker transcriptome of these cells, and validated the expression of five classical and nine non-classical markers.
Primary cell isolation and sample preparation for RNA analysis
Primary bone cells
Bone tissue was mechanically disrupted using a scalpel and resulting bone chips were plated onto tissue culture dishes in complete media [advanced minimum essential medium (MEM), 10 % phosphate-buffered saline (PBS), 100 U/ml penicillin, 100 g/ml streptomycin, 1x GlutaMAX] and maintained at 37 °C, 5 % CO2. Bone cells were plated into new culture dishes and passaged three times, at which time 1 × 106 cells were harvested for RNA analysis.
Human cartilage was first digested with 0.2 % pronase in complete media [Dulbecco’s modified Eagle’s medium [DMEM]/F12 10 % FBS, 100 U/ml penicillin, 100 g/ml streptomycin, 50 μg/mL gentamycin) for 1 h at 37 °C with shaking in a cell culture incubator. Following incubation with pronase, the cartilage was washed twice with PBS, then incubated with 0.036 % collagenase-P overnight at 37 °C in a cell culture incubator. The next day, undigested cartilage was removed using a cell strainer (BD Falcon) and flow-through containing primary chondrocytes was pelleted and washed twice with PBS. Primary chondrocytes were plated onto a tissue culture plate and maintained at 37 °C, 5 % CO2, after which 1 × 106 cells were harvested for RNA analysis.
Bone marrow-derived stromal cells
Primary human BMSC were purchased directly from Lonza and expanded using our previously described procedures . Cells were cultured up to passage 5 and plated on standard tissue culture plastic until 70–80 % confluent for harvest.
Primary human gingival fibroblasts (HGF) and human periodontal ligament (HPDL) cells were established from patient gingival connective tissue explants, as previously described . Cells were cultured at 37 °C in a 5 % CO2/95 % air atmosphere in DMEM containing 10 % FCS.
Real-time reverse transcriptase quantitative PCR analysis
Total RNA was isolated from primary cultured cells using either Trizol® Reagent (Thermo) or miRNeasy Mini Kit (Qiagen) according to respective protocols. The SuperScript III First-Strand Synthesis System (Invitrogen) was used to reverse transcribe RNA into cDNA, which was used as a template for real-time PCR analysis. Real-time reactions were performed with 10 ng cDNA per 10 μl with the QuantiTect SYBR Green PCR Kit (Qiagen) and detected using the CFX384 Real-Time System (BioRad). Gene expression levels were normalized to the housekeeping gene, GAPDH, and quantified using the 2^(−delta delta Ct) method. Gene specific primers are listed in Additional file 1: Table S1.
AMSC isolation and cell culture conditions
Mesenchymal stromal cells were derived from lipoaspirates obtained from consenting donors and clinical trial patients with approval from the Mayo Clinic Institutional Review Board (IRB) as previously described [12, 29]. After harvesting, fat tissue was digested with collagenase (type I at 0.075 %; Worthington Biochemicals) for 1.5 h at 37 °C. The stromal vascular fraction was isolated by low speed centrifugation (400 g for 5 min), the supernatant was removed, and the cell pellet was rinsed with PBS and passed through a cell strainer (70 μm) (BD Biosciences). Buffered ammonium solution (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) was used to lyse erythrocytes. The resulting cell fraction was plated onto tissue culture flasks in standard culture medium (advanced MEM) with 5 % hPL (Mill Creek Life Sciences), 2 mM L-glutamine (Invitrogen), and antibiotics (100 U/ml penicillin, 100 g/ml streptomycin) and maintained 37 °C in 5 % CO2 at a cell density of 1.0–2.5 × 103 cells/cm2.
Prior to cryopreservation, freshly isolated AMSCs were harvested for flow cytometric analysis (pre-thaw samples), and the remaining cells were frozen in 1 ml aliquots of up to 20 × 106 cells/mL with CryoStor CS10 Cryopreservation Medium (BioLife Solutions, Stem Cell Technologies) and stored in liquid nitrogen. Post-thaw samples were recovered from cryopreservation by placing the vial in a 37 °C water bath to thaw, then transferred to 10 ml of growth media and centrifuged for 5 min at 500 g. Cells were resuspended in growth media and approximately 1 × 106 cells were harvested for flow cytometric analysis. The remaining thawed cells were plated in T-175 cm2 flasks at a density of up to 3000 cells/cm2 and cultured for 4 days at 37 °C in 5 % CO2. After 4 days’ culture, cells were harvested for flow cytometric analysis.
List of antibodies used for flow cytometry validation
Alexa Fluor 647a
RNA-sequencing and bioinformatic analysis
Following RNA isolation using the miRNeasy Mini Kit (Qiagen), RNA-seq was performed as previously described . Briefly, RNA was prepared for sequencing using the TruSeq RNA Sample Prep Kit v2 (Illumina) and was analyzed using Illumina HiSeq 2000 with TruSeq SBS Kit v3 and HCS v2.0.12 data collection software. Sequence data were processed using MAPRSeq (v.1.2.1) and a bioinformatics workflow (TopHat 2.0.6, HTSeq, and edgeR 2.6.2), where expression data were normalized using the reads per kilobase per million (RPKM) method.
RNA-seq data were further analyzed using the DAVID Bioinformatics Resources 6.7 (https://david.ncifcrf.gov/) [30, 31]. Hierarchical clustering was performed using GENE-E (v3.0.228; Broad Institute, Cambridge, MA, USA). RNA-seq data were deposited in the public Gene Expression Omnibus (GEO) repository under the accession number [GEO:GSE84322].
Data were analyzed using GraphPad Prism v6 (GraphPad Software) and are presented as mean and individual data points. One-way ANOVA was used for multiple comparisons and post hoc analysis using Dunn’s multiple comparisons test was used to compare pre-freeze, post-thaw, and 4-day culture, where significance was set at p < 0.05.
AMSCs alter surface marker gene expression during proliferating or confluent culture conditions
Identification of non-classical cell surface markers of AMSCs and mesenchymal cells by high-throughput quantitative PCR screening
Evaluation of the qPCR panel across four clinical-grade AMSCs donors revealed 66/69 cell surface markers were detected. Of the cell surface markers analyzed, CD248 was the most highly expressed gene and demonstrated low co-variance between donor samples (data not shown). The surface marker qPCR panel was also used to analyze surface marker expression across other mesenchymal cell types, including BMSCs, fibroblasts, bone outgrowth cells, and primary digest chondrocytes. Hierarchical clustering analysis was used to compare AMSC surface markers to other cell types and revealed distinct clusters for the different cell types. This unsupervised analysis reveals that AMSCs form a unique cluster and that these cells are most similar to fibroblasts and BMSCs (Fig. 2b). The clustering analysis demonstrates that AMSCs are a distinct cell population and may be identified using gene expression profiling techniques for a panel of cell surface markers.
To develop a novel antibody panel that can distinguish AMSCs from other mesenchymal cell types, including fibroblasts, we compared surface marker gene expression between each lineage. Amongst the genes analyzed, CD36 was highly specific to AMSCs, whereas CD140B, CD271, and CD273 were able to discriminate AMSCs from BMSCs and fibroblasts (Fig. 2c). Compared to bone cells and chondrocytes, AMSCs did not express CD163, CD146, CD200, or CD274. In addition, CD248 was the cell surface marker most abundantly expressed by AMSCs. Taken together, these nine markers were able to distinguish AMSCs from other cell types. This demonstrates that qPCR is a useful technique for identifying informative markers for developing release criteria.
Flow cytometric validation of classical and non-classical surface markers in a cohort of clinical-grade AMSCs grown in human platelet lysate
Cell surface marker expression during various manufacturing conditions
We also evaluated the expression of non-classical markers across the three manufacturing time points (Fig. 5b). CD276 and CD140b were observed to exhibit a similar expression pattern to classical markers, as >90 % of gated cells were positive for these markers and were unchanged over the different conditions. Furthermore, the MFIs for these markers were significantly upregulated by day four after recovery from cryopreservation (Fig. 5b), which was also observed for the classical marker CD73. However, the non-classical markers generally demonstrated greater donor-to-donor variability compared to classical markers (Fig. 5b). In particular, CD271 and CD36 were the only markers analyzed that demonstrated a >20 % increase in the percentage of positive cells by day four after thawing, whereas CD44 and CD105 only increased by 2.43 % and 0.34 % respectively. The increased percentage of positive cells for CD271 and CD36 was also corroborated by a significant increase in MFI (Fig. 5b). Further studies are required to determine the biological importance of the increased abundance of CD271- and CD36-positive cells, but it may indicate positive selection of more robust cells or that recovery from cryopreservation is necessary for expression of growth factor receptors (CD271/NGFR) and fatty acid transporters (CD36). Collectively, these results suggest that cryopreservation does not modulate the expression of classical and non-classical cell surface markers. However, recovery in culture may allow optimal surface level expression of these proteins.
High-resolution surface marker gene expression analysis of clinical-grade AMSCs grown in human platelet lysate
During the manufacturing process AMSCs are expanded ex vivo to produce the large number of cells required for therapeutic applications. Throughout the process it is important to monitor the growth of the AMSCs and particularly the confluence of the cultures (surface area covered by cells), as these cells experience contact inhibition. To evaluate whether cell surface marker gene expression was modulated by the confluent state of the culture, RNA from proliferating (70–80 % confluent) and confluent (100 % confluent) cultures was analyzed. Gene expression analysis using the high-throughput qPCR panel developed above revealed 29 genes that were up-regulated greater than 2-fold in confluent cells and nine genes that were 2-fold up-regulated in proliferating cells. Classical surface markers CD44, CD105, and CD73 were not modulated by confluency; however, both CD90 and CD34 were >2-fold up-regulated in confluent cultures. Together, these results indicate that cell surface markers are directly modulated by the tissue culture conditions.
Top 25 categories from a DAVID 6.7 analysis of cell surface proteins expressed on adipose-derived mesenchymal stromal cells
(n = 142, p > 0.05)
(n = 104, up >1.4-fold, p < 0.05)
(n = 79, up >1.4-fold, p < 0.05)
Category (enrichment score)
Category (enrichment score)
Category (enrichment score)
Plasma membrane (28.33)
Cell adhesion (14.15)
Plasma membrane (11.82)
Cell adhesion (9.99)
Plasma membrane (12.38)
Signal transduction (8.17)
ABC transporter (3.93)
Growth factor binding (3.82)
Response to wounding (4.85)
Cell adhesion (3.39)
B cell activation/Fas pathway (5.08)
Immunoglobulin-like V set (4.57)
Cell migration (4.86)
Cell–cell adhesion (4.37)
Immune cell activation (2.55)
Immune response (4.38)
Vesicle-mediated transport (2.40)
Calcium-mediated signaling (4.20)
Cytokine–cytokine receptor interaction (2.33)
Cytokine binding (4.12)
Extracellular matrix-receptor interaction (3.37)
Cell migration (2.29)
Activation immune response (4.00)
Cell motion (3.36)
Cell motion (3.68)
Glycoprotein metabolic process (2.07)
Protein kinase cascade (3.64)
Epidermal growth factor (2.88)
Cell proliferation (1.96)
Low-density lipoprotein (2.32)
Membrane fraction (1.93)
Tyrosine protein kinase (3.04)
Biosynthetic process (2.20)
Immunoglobulin V-set (1.84)
Immune cell activation (3.02)
Regulation of transcription (1.56)
Immune response (2.11)
Response to wounding (1.48)
Low density lipoprotein receptor (2.91)
Membrane fraction (2.04)
Regulation of immune activation (1.38)
Stress-activated protein kinase (2.90)
Magnesium ion binding (1.92)
Protein kinase cascade (1.18)
Wnt receptor pathway (2.88)
Cytokine binding (1.75)
Carbohydrate binding (1.07)
Numerous clinical trials are currently being performed using AMSCs as a cellular therapy for various diseases worldwide [32, 33]. Standardization of the production procedures and accurate characterization of the MSC product to ensure patient safety has been a significant concern for regulatory agencies governing the approval of biological license applications . Our study identified and validated the expression of 14 classical and non-classical surface markers on clinical-grade AMSCs expanded in hPL adherent to good manufacturing practices (GMP-hPL). Furthermore, we evaluated surface marker expression during processes for preparing cells for clinical administration and demonstrated variability of these markers with doublings/day and cryopreservation.
Traditionally AMSCs have been expanded with FBS as part of the culture media to provide growth factors and other proteins to support proliferation. However, potential zoonotic pathogens and immunogenic reactions from FBS are concerning for the clinical administration of MSCs, which led to the development of nonzoonotic substitutes including hPL [12, 34]. As part of the production of clinical-grade AMSCs used in these studies, we expanded our MSC product in GMP-hPL, which has previously been shown to support proliferation and genomic stability . Furthermore, hPL contains proteins important for healing, including FGF/EGF, TGF-β/BMP, and VEGF/PDGF, which may facilitate AMSC growth and stability . However, due to the differences in composition of FBS and hPL, including cytokines and growth factors, there exists the potential for the selection of different adherent cell populations.
An AMSC cell population is characterized as one that adheres to plastic, expresses characteristic surface markers, and has tri-lineage potential . Establishing these criteria was an important step forward for standardization of stem cell science and industry. This study validated that clinical-grade AMSCs from 15 different donors met these established criteria and also expressed a unique set of non-classical surface markers. The AMSCs utilized in this investigation were cultured in GMP-hPL, adhered to plastic, and uniformly expressed the classical surface markers CD44, CD73, CD90, and CD105 and did not express CD34. Although these markers uniformly define AMSCs, ours and other studies have observed that these markers are unable to distinguish donor differences, including variability in proliferation or trophic activity . Therefore, there is an increased need to identify additional markers that not only define AMSCs but also have the potential to capture biological and manufacturing variability, as well as clinical performance.
Beyond serving as markers for cell characterization, surface proteins carry out important biological functions and are critical for cell-to-cell contact, extracellular matrix interactions, signal transduction, and transportation of molecules across the plasma membrane. Our study examined the expression of all plasma membrane protein-encoding genes (including CD markers, receptors, integrins, and transporters) by RNA-seq and identified 551/707 genes that were expressed on AMSCs. Previous studies of AMSCs and BMSCs evaluated the expression of 200–242 surface markers using mass spectroscopy or BD Lyoplate technology with flow cytometry techniques [21–24]. Together, our and others’ studies have identified and characterized a finite number of surface proteins present on the AMSC cell surface under standard conditions. A comparison of surface marker expression by qPCR expression and flow cytometry showed partial concordance, with seven out of nine markers showing similar trends (Fig. 4). Our results show that gene expression and flow cytometry techniques can be used to identify novel cell surface markers in AMSC populations. However, confirmation of cell surface proteins by flow cytometry is still necessary to confirm cell surface marker expression, as it is the primary technique used in the clinical setting.
Our data also reveal differential expression of mRNAs for CD markers in proliferative and post-proliferative AMSCs. Previous studies have also characterized the gene expression effects of confluence and doubling times on AMSCs and BMSCs [13, 37]. These studies and ours suggest that culture conditions, including proliferation state and population doublings, may affect the differentiation potential of AMSCs. In particular, mRNA expression of some surface markers is restricted to either the proliferating or confluent state (Fig. 6c). However, further studies are required to determine whether manufacturing conditions (e.g., length of culture and growth rate), as well as biological factors (e.g., donor age and disease status) would impact therapeutic potential. Our studies indicate that at least some variation in cell surface expression may emerge during AMSC production depending on the growth rate of the cell population.
Currently there is no standardization of release criteria for MSC products, and studies that define their criteria usually include the markers described by the ISCT and IFATS [2, 38]. Through the use of gene expression profiling techniques and flow cytometry, our study identified and validated nine non-classical markers that may help further characterize hPL-expanded AMSCs and improve current release criteria. In our study we validated the expression of CD163, CD271, CD200, CD36, CD274, CD146, CD248, CD140B, and CD276. These markers are also expressed on FBS-expanded AMSCs [22–25, 39–42]. Our study also identified CD163, a monocyte and macrophage marker, as a negative AMSC marker, which may be useful for characterizing clinical-grade MSC populations .
We also described the expression of the known immune-regulatory markers CD274 (B7H1/PD-L1) and CD276 (B7H3) on hPL-expanded AMSCs. Traditionally, mature dendritic cells produce soluble CD274 and CD276 . However, the current results demonstrate that AMSCs grown in hPL also express these markers on the cell surface. Our results show that CD276 is highly expressed and may be expressed ubiquitously with other traditional markers such as CD73, CD105, and CD90. Similarly, CD274 is highly expressed, but shows greater variability between donors. The functional role of CD274 and CD276 on AMSCs has yet to be characterized; however, CD274-positive BMSCs have been shown to regulate T-cell proliferation and Th17 polarization [45, 46]. Furthermore, recent studies have shown that interferon gamma (IFNγ) priming or licensing of BMSCs may also up-regulate CD274 and enhance MSC-mediated T-cell inhibition [47, 48]. However, the function of CD276 remains controversial as this molecule may act as a co-stimulatory molecule for T-cell activation and selectively stimulates the production of IFNγ , or may inhibit T-cell proliferation . CD274 and CD276 have the potential to serve as predictive clinical markers for MSC immunomodulatory activity. Isolation and characterization of CD274-positive and CD276-positive cells may determine whether these cells represent distinct subpopulations with enhanced immune-regulatory effects. Further studies correlating these markers with patient outcomes in clinical trials would also help to elucidate the role of these markers in hPL-expanded AMSCs.
Storage and administration of the MSC product for cell therapy may depend on the disease and the institutional infrastructure. Current clinical trials administer MSC products either without cryopreservation, cryopreserved and thawed, or allowed to recover for 4 days in culture. Previous studies have demonstrated reduced immunosuppressive properties of MSCs immediately thawed after cryopreservation, and that these properties were restored as early as 24 h after placing in tissue culture . Samples from three of our five donors analyzed showed a slight decrease in CD248 expression between pre-freeze and post-thaw samples. The decrease in surface marker expression could be attributed to damage to the cell surface of the protein that reduces antibody-binding efficiency or, potentially, the sensitivity of CD248 expression to the metabolic state of the cell. We also observed a significant increase in CD105 expression between pre-freeze and post-thaw samples, as well as a significant increase in surface marker expression over 4 days for CD271, CD36, and, to a lesser extent, CD44. These results support the work of Francois and colleagues , whereby the recovery of AMSCs in culture for up to 4 days can result in maximal surface marker expression. Together, these data suggest that surface marker expression is modulated during the cryopreservation process and that it may be important for cell function to allow cells to recover for up to 4 days.
We evaluated the expression of classical and non-classical surface markers across a cohort of 15 donors from various disease backgrounds. Our results demonstrate that our manufacturing procedures consistently produce an AMSC population that uniformly expresses classical MSC surface markers. We also characterized the expression of nine non-classical surface markers that may be used to further characterize the AMSC product. The known immunomodulatory markers CD274 and CD276 are also highly expressed on the surface of AMSCs and may be able to predict their immunomodulatory activity and clinical efficacy. Future clinical trials will help us to determine which surface markers are the best predictors of clinical outcomes for patients.
ALS, Amyotrophic lateral sclerosis; AMSC, Adipose derived mesenchymal stromal cell; ARAS , Atherosclerotic renal artery stenosis; BMSC, Bone marrow derived mesenchymal stromal cell; DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, Fetal bovine serum; FCS, Fetal calf serum; FDA, Food and drug administration; Geo, Geometric Mean; GMP, Good manufacturing practices; HGF, human gingival fibroblasts; HPDL, human periodontal ligament; hPL, Human platelet lysate; IFATS, International Federation of Adipose Therapeutics and Sciences; IRB, Institutional Review Board; ISCT, International Society for Cellular Therapy; MFI, Mean fluorescence intensity; MSA, Multiple systems atrophy; MSC, Mesenchymal stromal cell; PBS, phosphate-buffered saline; qPCR, quantitative polymerase chain reaction; RNA-seq, RNA sequencing; RPKM, Reads per kilobase per million mapped reads
We thank the members of our research groups (Xiaodong Li) and the Human Cell Therapy laboratory (Jarrett Anderson, Greg Butler, Darcie J. Radel, Peggy Bulur) at Mayo Clinic.
This work was supported by intramural funds from the Center of Regenerative Medicine at Mayo Clinic and NIH R01 grants AR049069 (to AJvW), F32 AR066508 (AD), and a Mayo Clinic Centre for Regenerative Medicine fellowship (to SMR). We also appreciate the generous philanthropic support of William H. and Karen J. Eby, and the charitable foundation in their name.
Availability of data and materials
The RNA-seq data generated and analyzed in the current study are available in the Gene Expression Omnibus (GEO) database with the accession number [GEO:GSE84322].
ETC, MPG, AD, SMR, CGG, CRP, and HT contributed to the study design, study performance, data collection, and preparation of the manuscript. MK contributed to data analysis and interpretation, and revision of the manuscript. SC and HIS contributed to the study performance, data analysis and interpretation, and revision of the manuscript. ANL, WQ, and JS helped with the revision and approval of the final manuscript. ABD and AJvW contributed to the study design, interpretation, and revision of the manuscript. All authors read and approved the final manuscript.
The authors declare the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: ABD and MPG have commercial interest in Mill Creek Life Sciences, which manufactures the clinical-grade commercial platelet lysate product used for maintaining adipose tissue-derived mesenchymal stromal cells.
Consent for publication
Ethics approval and consent to participate
Lipoaspirates and tissues were obtained from consenting donors and clinical trial patients with approval from the Mayo Clinic Institutional Review Board.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Kaplan JM, Youd ME, Lodie TA. Immunomodulatory activity of mesenchymal stem cells. Curr Stem Cell Res Ther. 2011;6(4):297–316.View ArticlePubMedGoogle Scholar
- Mendicino M, Bailey AM, Wonnacott K, Puri RK, Bauer SR. MSC-based product characterization for clinical trials: an FDA perspective. Cell Stem Cell. 2014;14(2):141–5. doi:10.1016/j.stem.2014.01.013.View ArticlePubMedGoogle Scholar
- Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC) [database on the Internet]. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Available from: https://clinicaltrials.gov/ct2/show/NCT01931007. Accessed: 16 Nov 2015.
- MSC for Occlusive Disease of the Kidney [database on the Internet]. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Available from: https://clinicaltrials.gov/ct2/show/NCT01840540. Accessed: 16 Nov 2015.
- Mesenchymal Stem Cell Therapy in Multiple System Atrophy. [database on the Internet]. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Available from: https://clinicaltrials.gov/ct2/show/NCT02315027. Accessed: 16 Nov 2015.
- Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. [database on the Internet]. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Available from: https://clinicaltrials.gov/ct2/show/NCT00366145. Accessed: 16 Nov 2015.
- A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. [database on the Internet]. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Available from: https://clinicaltrials.gov/ct2/show/NCT01609283. Accessed: 16 Nov 2015.
- Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143–7.View ArticlePubMedGoogle Scholar
- De Ugarte DA, Morizono K, Elbarbary A, Alfonso Z, Zuk PA, Zhu M, et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs. 2003;174(3):101–9. doi:10.1159/000071150.View ArticlePubMedGoogle Scholar
- Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7(2):211–28. doi:10.1089/107632701300062859.View ArticlePubMedGoogle Scholar
- Bourin P, Bunnell BA, Casteilla L, Dominici M, Katz AJ, March KL, et al. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy. 2013;15(6):641–8. doi:10.1016/j.jcyt.2013.02.006.View ArticlePubMedPubMed CentralGoogle Scholar
- Crespo-Diaz R, Behfar A, Butler GW, Padley DJ, Sarr MG, Bartunek J, et al. Platelet lysate consisting of a natural repair proteome supports human mesenchymal stem cell proliferation and chromosomal stability. Cell Transplant. 2011;20(6):797–811. doi:10.3727/096368910X543376.View ArticlePubMedGoogle Scholar
- Dudakovic A, Camilleri E, Riester SM, Lewallen EA, Kvasha S, Chen X, et al. High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells. J Cell Biochem. 2014;115(10):1816–28. doi:10.1002/jcb.24852.View ArticlePubMedPubMed CentralGoogle Scholar
- Jurgens WJ, Oedayrajsingh-Varma MJ, Helder MN, Zandiehdoulabi B, Schouten TE, Kuik DJ, et al. Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies. Cell Tissue Res. 2008;332(3):415–26. doi:10.1007/s00441-007-0555-7.View ArticlePubMedPubMed CentralGoogle Scholar
- Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M, Helder MN, Klein-Nulend J, Schouten TE, et al. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Cytotherapy. 2006;8(2):166–77. doi:10.1080/14653240600621125.View ArticlePubMedGoogle Scholar
- Ho AD, Wagner W, Franke W. Heterogeneity of mesenchymal stromal cell preparations. Cytotherapy. 2008;10(4):320–30. doi:10.1080/14653240802217011.View ArticlePubMedGoogle Scholar
- Cholewa D, Stiehl T, Schellenberg A, Bokermann G, Joussen S, Koch C, et al. Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density. Cell Transplant. 2011;20(9):1409–22. doi:10.3727/096368910X557218.View ArticlePubMedGoogle Scholar
- Song IH, Caplan AI, Dennis JE. Dexamethasone inhibition of confluence-induced apoptosis in human mesenchymal stem cells. J Orthop Res. 2009;27(2):216–21. doi:10.1002/jor.20726.View ArticlePubMedGoogle Scholar
- Davies OG, Smith AJ, Cooper PR, Shelton RM, Scheven BA. The effects of cryopreservation on cells isolated from adipose, bone marrow and dental pulp tissues. Cryobiology. 2014;69(2):342–7. doi:10.1016/j.cryobiol.2014.08.003.View ArticlePubMedGoogle Scholar
- Marquez-Curtis LA, Janowska-Wieczorek A, McGann LE, Elliott JA. Mesenchymal stromal cells derived from various tissues: biological, clinical and cryopreservation aspects. Cryobiology. 2015;71(2):181–97. doi:10.1016/j.cryobiol.2015.07.003.View ArticlePubMedGoogle Scholar
- Niehage C, Steenblock C, Pursche T, Bornhauser M, Corbeil D, Hoflack B. The cell surface proteome of human mesenchymal stromal cells. PLoS One. 2011;6(5):e20399. doi:10.1371/journal.pone.0020399.View ArticlePubMedPubMed CentralGoogle Scholar
- Donnenberg AD, Meyer EM, Rubin JP, Donnenberg VS. The cell-surface proteome of cultured adipose stromal cells. Cytometry A. 2015;87(7):665–74. doi:10.1002/cyto.a.22682.View ArticlePubMedGoogle Scholar
- Walmsley GG, Atashroo DA, Maan ZN, Hu MS, Zielins ER, Tsai JM, et al. High-throughput screening of surface marker expression on undifferentiated and differentiated human adipose-derived stromal cells. Tissue Eng Part A. 2015;21(15–16):2281–91. doi:10.1089/ten.TEA.2015.0039.View ArticlePubMedPubMed CentralGoogle Scholar
- Baer PC, Kuci S, Krause M, Kuci Z, Zielen S, Geiger H, et al. Comprehensive phenotypic characterization of human adipose-derived stromal/stem cells and their subsets by a high throughput technology. Stem Cells Dev. 2013;22(2):330–9. doi:10.1089/scd.2012.0346.View ArticlePubMedGoogle Scholar
- Astori G, Vignati F, Bardelli S, Tubio M, Gola M, Albertini V, et al. "In vitro" and multicolor phenotypic characterization of cell subpopulations identified in fresh human adipose tissue stromal vascular fraction and in the derived mesenchymal stem cells. J Transl Med. 2007;5:55. doi:10.1186/1479-5876-5-55.View ArticlePubMedPubMed CentralGoogle Scholar
- Zimmerlin L, Donnenberg VS, Rubin JP, Donnenberg AD. Mesenchymal markers on human adipose stem/progenitor cells. Cytometry A. 2013;83(1):134–40. doi:10.1002/cyto.a.22227.View ArticlePubMedGoogle Scholar
- Helledie T, Dombrowski C, Rai B, Lim ZX, Hin IL, Rider DA, et al. Heparan sulfate enhances the self-renewal and therapeutic potential of mesenchymal stem cells from human adult bone marrow. Stem Cells Dev. 2012;21(11):1897–910. doi:10.1089/scd.2011.0367.View ArticlePubMedGoogle Scholar
- Ogata Y, Niisato N, Sakurai T, Furuyama S, Sugiya H. Comparison of the characteristics of human gingival fibroblasts and periodontal ligament cells. J Periodontol. 1995;66(12):1025–31. doi:10.1902/jop.19126.96.36.1995.View ArticlePubMedGoogle Scholar
- Mader EK, Butler G, Dowdy SC, Mariani A, Knutson KL, Federspiel MJ, et al. Optimizing patient derived mesenchymal stem cells as virus carriers for a phase I clinical trial in ovarian cancer. J Transl Med. 2013;11:20. doi:10.1186/1479-5876-11-20.View ArticlePubMedPubMed CentralGoogle Scholar
- da Huang W, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37(1):1–13. doi:10.1093/nar/gkn923.View ArticleGoogle Scholar
- da Huang W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44–57. doi:10.1038/nprot.2008.211.View ArticleGoogle Scholar
- Dave M, Mehta K, Luther J, Baruah A, Dietz AB, Faubion Jr WA. Mesenchymal stem cell therapy for inflammatory bowel disease: a systematic review and meta-analysis. Inflamm Bowel Dis. 2015;21(11):2696–707. doi:10.1097/MIB.0000000000000543.View ArticlePubMedGoogle Scholar
- Chen BK, Staff NP, Knight AM, Nesbitt JJ, Butler GW, Padley DJ, et al. A safety study on intrathecal delivery of autologous mesenchymal stromal cells in rabbits directly supporting Phase I human trials. Transfusion. 2015;55(5):1013–20. doi:10.1111/trf.12938.View ArticlePubMedGoogle Scholar
- Muller I, Kordowich S, Holzwarth C, Spano C, Isensee G, Staiber A, et al. Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM. Cytotherapy. 2006;8(5):437–44. doi:10.1080/14653240600920782.View ArticlePubMedGoogle Scholar
- Ng F, Boucher S, Koh S, Sastry KS, Chase L, Lakshmipathy U, et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood. 2008;112(2):295–307. doi:10.1182/blood-2007-07-103697.View ArticlePubMedGoogle Scholar
- Zhang M, Mal N, Kiedrowski M, Chacko M, Askari AT, Popovic ZB, et al. SDF-1 expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction. FASEB J. 2007;21(12):3197–207. doi:10.1096/fj.06-6558com.View ArticlePubMedGoogle Scholar
- Okolicsanyi RK, Camilleri ET, Oikari LE, Yu C, Cool SM, van Wijnen AJ, et al. Human mesenchymal stem cells retain multilineage differentiation capacity including neural marker expression after extended in vitro expansion. PLoS One. 2015;10(9):e0137255. doi:10.1371/journal.pone.0137255.View ArticlePubMedPubMed CentralGoogle Scholar
- Robey PG, Kuznetsov SA, Ren J, Klein HG, Sabatino M, Stroncek DF. Generation of clinical grade human bone marrow stromal cells for use in bone regeneration. Bone. 2015;70:87–92. doi:10.1016/j.bone.2014.07.020.View ArticlePubMedGoogle Scholar
- Calabrese G, Giuffrida R, Lo Furno D, Parrinello NL, Forte S, Gulino R, et al. Potential effect of CD271 on human mesenchymal stromal cell proliferation and differentiation. Int J Mol Sci. 2015;16(7):15609–24. doi:10.3390/ijms160715609.View ArticlePubMedPubMed CentralGoogle Scholar
- Braun J, Kurtz A, Barutcu N, Bodo J, Thiel A, Dong J. Concerted regulation of CD34 and CD105 accompanies mesenchymal stromal cell derivation from human adventitial stromal cell. Stem Cells Dev. 2013;22(5):815–27. doi:10.1089/scd.2012.0263.View ArticlePubMedGoogle Scholar
- Rider DA, Dombrowski C, Sawyer AA, Ng GH, Leong D, Hutmacher DW, et al. Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells. Stem Cells. 2008;26(6):1598–608. doi:10.1634/stemcells.2007-0480.View ArticlePubMedGoogle Scholar
- Zannettino AC, Paton S, Arthur A, Khor F, Itescu S, Gimble JM, et al. Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo. J Cell Physiol. 2008;214(2):413–21. doi:10.1002/jcp.21210.View ArticlePubMedGoogle Scholar
- Van den Heuvel MM, Tensen CP, van As JH, Van den Berg TK, Fluitsma DM, Dijkstra CD, et al. Regulation of CD 163 on human macrophages: cross-linking of CD163 induces signaling and activation. J Leukoc Biol. 1999;66(5):858–66.PubMedGoogle Scholar
- Frigola X, Inman BA, Krco CJ, Liu X, Harrington SM, Bulur PA, et al. Soluble B7-H1: differences in production between dendritic cells and T cells. Immunol Lett. 2012;142(1–2):78–82. doi:10.1016/j.imlet.2011.11.001.View ArticlePubMedGoogle Scholar
- Yan Z, Zhuansun Y, Liu G, Chen R, Li J, Ran P. Mesenchymal stem cells suppress T cells by inducing apoptosis and through PD-1/B7-H1 interactions. Immunol Lett. 2014;162(1 Pt A):248–55. doi:10.1016/j.imlet.2014.09.013.View ArticlePubMedGoogle Scholar
- Luz-Crawford P, Noel D, Fernandez X, Khoury M, Figueroa F, Carrion F, et al. Mesenchymal stem cells repress Th17 molecular program through the PD-1 pathway. PLoS One. 2012;7(9):e45272. doi:10.1371/journal.pone.0045272.View ArticlePubMedPubMed CentralGoogle Scholar
- Sheng H, Wang Y, Jin Y, Zhang Q, Zhang Y, Wang L, et al. A critical role of IFNgamma in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1. Cell Res. 2008;18(8):846–57. doi:10.1038/cr.2008.80.View ArticlePubMedGoogle Scholar
- Chinnadurai R, Copland IB, Patel SR, Galipeau J. IDO-independent suppression of T cell effector function by IFN-gamma-licensed human mesenchymal stromal cells. J Immunol. 2014;192(4):1491–501. doi:10.4049/jimmunol.1301828.View ArticlePubMedGoogle Scholar
- Chapoval AI, Ni J, Lau JS, Wilcox RA, Flies DB, Liu D, et al. B7-H3: a costimulatory molecule for T cell activation and IFN-gamma production. Nat Immunol. 2001;2(3):269–74. doi:10.1038/85339.View ArticlePubMedGoogle Scholar
- Suh WK, Gajewska BU, Okada H, Gronski MA, Bertram EM, Dawicki W, et al. The B7 family member B7-H3 preferentially down-regulates T helper type 1-mediated immune responses. Nat Immunol. 2003;4(9):899–906. doi:10.1038/ni967.View ArticlePubMedGoogle Scholar
- Francois M, Copland IB, Yuan S, Romieu-Mourez R, Waller EK, Galipeau J. Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-gamma licensing. Cytotherapy. 2012;14(2):147–52. doi:10.3109/14653249.2011.623691.View ArticlePubMedGoogle Scholar