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Mar 26, 2019

ORS 2019 Annual Meeting

2198 - Bone Marrow Aspirate Concentrate (BMAC) as a Source of Mesenchymal Stem Cells (MSCs). A Single-Cell Transcriptomics Study

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progenitor and stem cells

mesenchymal

bmac

bone marrow aspirate

Abstract

Abstract

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Keywords

progenitor and stem cells

mesenchymal

bmac

bone marrow aspirate

Abstract

Bone marrow aspirate concentrate (BMAC) is becoming a more common regenerative medicine source tissue in orthopaedic surgery. Studies have demonstrated clinical benefits of both BMAC and derived mesenchymal stem cells (MSCs) in multiple clinical scenarios, including treatment of arthritis, pain, and regeneration of cartilage (1-7) though its use in these conditions is not FDA approved. BMAC is aspirated autologous bone marrow that is fractionated through centrifugation in order to theoretically concentrate MSCs and limit other viable cell types. MSCs are multi-potent progenitor cells capable of differentiating along a tri-lineage pathway of osteoblasts, chondroblasts, or adipocytes. There are several surface marker-based guidelines for identifying these cells, and although gene expression methods have been proposed, they are not widely accepted or reproduced in the literature (8-11). In one study, flow cytometry was used to quantify MSCs, which were reported to account for 0.01-0.001% of human bone marrow when taken from the iliac crest (12). The relationship between these surface markers and the multipotency of the cells is the focus of contemporary debate in the literature, with some groups believing that MSCs actually represent multiple, more differentiated cell types. Purpose of this study: to quantify the number of MSCs present in human bone marrow harvested from the iliac crest using a single cell transciptomics approach, which would in future allow us to explore the potential heterogeneity of this debated cell population. A total of 11 adult subjects were included and underwent bone marrow aspiration in addition to their orthopaedic surgery. The average age of the patients was 46.2 years (25-68), 5 patients were male (56%), 4 patients were females (44%), with average BMI 24.8 (21.2-30.1) for men and 24.9 (19.5-31.7) for women. A total of 60 mL of bone marrow was aspirated from the iliac crest and centrifuged using the Arthrex Angel System to yield approximately 2-5 mL of BMAC, of which 1 mL was used for single cell RNA sequencing. BMAC samples initially underwent processing to obtain a single cell suspension, followed by standard processing (14) to generate individually barcoded cDNA for amplification and library construction(10X) with subsequent transcriptome sequencing performed by HISEQ400 (Illumina). Each patient’s individual cell transcriptomes were evaluated for the presence of expected hematopoietic cells and MSC’s using published computational biology data and methods (8-11). Two patient samples were excluded because of poor RNA yields. A total of 12,850 cells were analyzed from 9 patients with an average of 1427 (609-2600) per patient BMAC sample. Analysis utilizing previously published strict identification methods with surface markers revealed MSCs represented 0.00 – 0.08% of BMAC cells (Table 1). After adjusting strict surface marker and gene transcript definitions to a more liberal method of identification, MSCs represented 0.15 – 2.78% of BMAC cells (Table 1). Interestingly, there was very little overlap in the individual cells identified as MSCs using the 5 different definitions (Figure 1). Our data indicates that the strict surface marker or gene expression definitions yield minimal to no MSCs in patient BMAC samples. This may be the result of prior definitions being generated from aggregate instead of individual cell data, and is consistent with some prior work (13). When these definitions are adjusted to be more liberal, the average fractions of MSCs is 0.15-2.78% per patient sample. The obvious follow-up questions are: 1) are these more liberally defined cells multipotent?, and 2) do they follow a differentiation pathway consistent with an MSC? It is clear from a transcriptional perspective that MSCs, regardless of the definition used, appear to be quite heterogeneous. Our study is the first to quantify MSCs in human BMAC harvested from the iliac crest using single-cell transcriptional profiling. Clinically, these data raise important questions about BMAC as a potential source of MSCs, and if so, their subtypes and potency (in vivo and in vitro). Current work should exploit transcriptional data to isolate marrow-derived cell subpopulations and challenge their efficacy with the long-term goal of determining the proportion of MSCs (or any cell type) needed to have a tissue-level effect, ultimately leading to a clinical effect. 1. Afizah, H.J. Clinical Ortho Trauma, 2016, 2. Shapiro S. AJSM 2016, 3. Imam M., Int. Ortho 2017, 4. Jager M., Curr Stem Cell Research 2009, 5. Gobbi A, Cartilage 2009, 6. Vangsness C, JBJS 2014, 7. Chalha J., OJSM 2016, 8. Ghazanfari R. Nature Sci Rep, 2017, 9. Dominici Cytotherapy, 2006, 10. Jia L., Genomics 2002, 11. Silva, Stem Cells 2003, 12. Pittenger M. Science, 1999, 13. Robinson, R. AJSM 2018 14. Satija, R., Nat Biotechnol 2015

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© Copyright 2019 Morressier GmbH.
All rights reserved.