Molecular Phenotyping of Telomerized Human Bone Marrow Skeletal Stem Cells Reveals a Genetic Program of Enhanced Proliferation and Maintenance of Differentiation Responses.

Natalie A Twine,Moustapha Kassem,Abdullah Aldahmash,James Adjaye,Marc R Wilkins,Linda Harkness

doi:10.1002/jbm4.10050

Abstract

ABSTRACTLong‐term in vitro expansion of bone marrow stromal (skeletal) stem cells (also known as human mesenchymal stem cells [hMSC]) is associated with replicative senescence and impaired functions. We have previously reported that telomerization of hMSC through hTERT overexpression led to bypassing a replicative senescence phenotype and improved in vitro and in vivo functions. However, the molecular consequence of telomerization is poorly characterized. Thus, we compared the molecular phenotype of a well‐studied telomerized hMSC (hMSC‐TERT) cell line with primary hMSC. At a cellular level, both cell populations exhibited strong concordance for the known hMSC CD markers, similar responses to osteoblast (OB) differentiation induction, and formed heterotopic bone in vivo. Overall gene expression was highly correlated between both cell types with an average Pearson's correlation coefficient (R2) between the gene expression of all primary hMSC and all hMSC‐TERT samples of 0.95 (range 0.93–0.96). Quantitative analysis of gene expression of CD markers, OB cell markers, and transcription factors (TF) showed a high degree of similarity between the two cell populations (72%, 77%, and 81%, respectively). The hMSC‐TERT population was enriched mainly for genes associated with cell cycle and cell cycle signaling when compared with primary hMSC. Other enrichment was observed for genes involved in cell adhesion and skeletal system development and immune response pathways. Interestingly, hMSC‐TERT shared a telomerization signature with upregulation of cancer/testis antigens, MAGE, and PAGE genes. Our data demonstrate that the enhanced biological characteristics of hMSC after telomerization are mainly due to enhanced expression of cell proliferation genes, whereas gene expression responses to differentiation are maintained. © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research

Highlights

IntroductionHuman bone marrow-derived skeletal ( known as stromal or mesenchymal stem cells; hMSC) are multipotent adult stem cells
Human bone marrow-derived skeletal are multipotent adult stem cells
There was a similar percentage of positive cells for CD44, CD63, CD73, CD90, CD105, CD146, and CD166, between primary hMSC and hMSC-TERT cells. Both cell populations were negative for CD14, which is a known marker for monocytes and macrophages.[28]. Primary hMSC and hMSCTERT showed similar morphologies (Fig. 1B)

Summary

Introduction

Human bone marrow-derived skeletal ( known as stromal or mesenchymal stem cells; hMSC) are multipotent adult stem cells. They are present in the bone marrow stroma and capable of self-renewal and multi-lineage differentiation into mesoderm-type cells such as osteoblasts, adipocytes, and chondrocytes.[1,2] In addition, hMSC exhibit immune-modulatory and regeneration-enhancing characteristics based on secretion of a large number of molecules.[3] These qualities have encouraged clinical testing of hMSC for the enhancement of tissue regeneration after injury. Global molecular phenotyping techniques such as DNA microarrays can be used This method has been applied to the unbiased classification of cancer subtypes, as described in the landmark study by Golub and colleagues,(19) but it has not been widely used within the hMSC field. We investigated molecular phenotype by examining a set of osteoblast (OB) gene markers that are differentiation stagespecific,(20) along with genes for cell surface markers, transcription factors, immune molecules, and signaling pathways

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Results

Conclusion