Human Mesenchymal Stromal Cell (MSC) Characteristics Vary Among Laboratories When Manufactured From the Same Source Material: A Report by the Cellular Therapy Team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative.

David F Stroncek,Ping Jin,Shibani Pati,Minoko Takanashi,Emily Peterson,Magali J Fontaine,Jo-Anna Reems,Eric Benedetti,Richard Schäfer,David H Mckenna

doi:10.3389/fcell.2020.00458

Abstract

BackgroundCulture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods and different source materials. The purpose of this study was to assess the impact on MSC characteristics when different laboratories propagated MSCs from cultures initiated with BM aliquots derived from the same donor source material.Methods and MethodsFive aliquots from each of three different BM donors were distributed to five independent laboratories. Three laboratories plated whole BM and two laboratories a mononuclear BM cell fraction. Four laboratories cultured in media supplemented with fetal bovine serum (FBS) and one laboratory used human platelet lysate (hPL). Initial cell seeding densities (i.e., P0) ranged from 19.7 × 103/cm2–282 × 103/cm2 and for second seeding (i.e., P1) 0.05 × 103–5.1 × 103 cells/cm2. Post-thawed MSCs from each laboratory were analyzed for cell viability, immunophenotype, tri-lineage differentiation, fibroblast colony-forming units (CFU-F), gene expression, and immunosuppressive activity.ResultsTransit times from BM collection to receipt by laboratories located in the United States ranged from 16.0–30.0 h and from 41.5–71.5 h for a laboratory in Asia. Post-thaw culture derived MSCs rom BM #1, #2, and #3 exhibited viabilities that ranged from 74–92%, 61–96%, and 23–90%, respectively. CFU activity from BM #1, #2, and #3 per 200 MSCs plated averaged 45.1 ± 21.4, 49.3 ± 26.8 and 14.9 ± 13.3, respectively. No substantial differences were observed in immunophenotype, and immunosuppressive activities. Global gene expression profiles of MSCs revealed transcriptome differences due to different inter-laboratory methods and to donor source material with the center effects showing greater molecular differences than source material.ConclusionFunctional and molecular differences exist among MSCs produced by different centers even when the same BM starting material is used to initiate cultures. These results indicated that manufacturing of MSCs by five independent centers contributed more to MSC variability than did the source material of the BM used in this study. Thus, emphasizing the importance of establishing worldwide standards to propagate MSCs for clinical use.

Highlights

mesenchymal stromal cell (MSC) are a diverse population of cells that are under investigation for the treatment of a wide range of diseases and disorders that include graft-versus-host disease (GvHD) (Le Blanc et al, 2008), stroke, (Lalu et al, 2019) Crohn’s disease (Forbes, 2017), osteogenesis imperfect (Horwitz et al, 1999), osteoarthritis, (Migliorini et al, 2019) multiple sclerosis (Uccelli et al, 2019), and cardiovascular disease (Yun and Lee, 2019)
The discrepancies found in the effectiveness of using MSCs in clinical studies may be Abbreviations: 7-AAD, 7-aminoactinomycin D; APC, allophycocyanin; Best, Biomedical Excellence in Safer Transfusion; BM, bone marrow; CFU-F, colony forming unit-fibroblast; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GvHD, graft-versus-host disease; hPL, human platelet lysate; LN2, liquid nitrogen; MLR, mixed lymphocyte reaction; MNC, mononuclear cell; MSC, mesenchymal stromal cell; P#, passage number; Principal Component Analysis (PCA), principal component analysis; PD, population doubling; PE, phycoerythrin; RCT, randomized control trials
We report that inter-laboratory manufacturing differences make a greater contribution to MSC variability than the differences noted among the BM donors utilized in this study

Summary

Introduction

MSCs are a diverse population of cells that are under investigation for the treatment of a wide range of diseases and disorders that include graft-versus-host disease (GvHD) (Le Blanc et al, 2008), stroke, (Lalu et al, 2019) Crohn’s disease (Forbes, 2017), osteogenesis imperfect (Horwitz et al, 1999), osteoarthritis, (Migliorini et al, 2019) multiple sclerosis (Uccelli et al, 2019), and cardiovascular disease (Yun and Lee, 2019). A recent comprehensive review of completed randomized clinical trials (RCTs) that used MSCs for the treatment of GvHD found that MSCs might have little or no effect (Fisher et al, 2019). The discrepancies found in the effectiveness of using MSCs in clinical studies may be Abbreviations: 7-AAD, 7-aminoactinomycin D; APC, allophycocyanin; Best, Biomedical Excellence in Safer Transfusion; BM, bone marrow; CFU-F, colony forming unit-fibroblast; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GvHD, graft-versus-host disease; hPL, human platelet lysate; LN2, liquid nitrogen; MLR, mixed lymphocyte reaction; MNC, mononuclear cell; MSC, mesenchymal stromal cell; P#, passage number; PCA, principal component analysis; PD, population doubling; PE, phycoerythrin; RCT, randomized control trials. The purpose of this study was to assess the impact on MSC characteristics when different laboratories propagated MSCs from cultures initiated with BM aliquots derived from the same donor source material

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