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

Abstract

BackgroundClinical 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).MethodsIn 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.ResultsWe 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.ConclusionsOur 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.Clinical trialsUse 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0370-8) contains supplementary material, which is available to authorized users.

Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing

“…Platelet-lysate expanded MSCs derived from the stromal vascular fraction of adipose-tissue are `Good Manufacturing Practice'-compliant pericyte-like immature fibroblasts that are used in clinical trials, have multi-lineage potential and express all relevant markers expected of mesenchymal stem cells [Camilleri et al, 2016; Dudakovic et al, 2014; Dudakovic et al, 2015a; Riester et al, 2016]. MSCs were harvested from lipo-aspirates obtained from consenting healthy donors as previously described [Crespo-Diaz et al, 2011; Mader et al, 2013] with approval from the Mayo Clinic Institutional Review Board.…”

“…High throughput next generation RNA-sequencing (RNA-seq) of polyA mRNAs and bioinformatic analyses were performed as previously reported [Camilleri et al, 2016; Dudakovic et al, 2014]. Gene expression is expressed in reads per kilobasepair per million mapped reads (RPKM) and are accessible through the NCBI Gene Expression Omnibus using series accession number GSE84322.…”

Adipose, Bone Marrow and Synovial Joint-Derived Mesenchymal Stem Cells for Cartilage Repair

“…However, ASCs also exhibit various other cell markers which are associated with stemness such as POU5F1, NANOG and KLF4 (Dudakovic et al, 2014; Lv et al, 2014; Camilleri et al, 2016). ASCs may exhibit individual variations of these marker patterns amongst patients or depending on the locations of the source-fat deposit (i.e., subcutaneous or visceral) in the body (Yang et al, 2014).…”

Comparison of human platelet lysate alternatives using expired and freshly isolated platelet concentrates for adipose-derived stromal cell expansion

“…These properties have resulted in the use of ASCs for cellular therapies, as evidenced by the fact that their therapeutic effect is being assessed in over 180 clinical trials (2,3). Of those clinical trials that have listed their expansion conditions and media preparation (4), the majority make use of foetal bovine serum (FBS), which has had adverse reactions on patients receiving the cells (4).…”

“…It has therefore been suggested that the choice of human alternative should be determined on the basis of the proposed downstream application of the ASCs (8,(15)(16)(17). Human platelet lysate (HPL) has been used in the past few years as an alternative to FBS for GMP-compliant ASC expansion and is being used in several clinical trials (3,15,18,19). Both autologous (recipient-derived) and allogeneic (donor-derived) HPL have been investigated for ASC expansion with favourable outcomes (20,21).…”