Degree

Doctor of Philosophy (PhD)

Department

Comparative Biomedical Sciences

Document Type

Dissertation

Abstract

Human mesenchymal stem cells (hMSCs) are the workhorse of regenerative medicine; large quantities of hMSCs obtained via in vitro expansion prior to transplantation are required to meet therapeutic purposes. However, hMSCs quickly lose their osteogenic differentiation potential during in vitro expansion, which is a major roadblock to their clinical applications. This dissertation aims to understand the molecular mechanisms underlying this in vitro expansion-related loss of osteogenic capacity in hMSCs. By comparing the transcriptome changes following in vitro expansion of hMSCs, cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) was identified as the most downregulated gene, which further validated by Western blotting showing that both the secreted and non-secreted CRISPLD2 proteins progressively declined in hMSCs during in vitro expansion when the cells gradually lost their osteogenic potential. Knockdown of CRISPLD2 in early passage hMSCs inhibited the cells’ osteogenic differentiation, which might be attributed to the downregulation of pro-osteogenic regulators matrix metallopeptidase 1 (MMP1) and forkhead box Q1 (FOXQ1). Furthermore, adeno-associated virus (AAV)-mediated CRISPLD2 overexpression could rescue the impaired osteogenic differentiation of human bone marrow stem cells (hBMSCs) during in vitro expansion. Given the pro-osteogenic effect of CRISPLD2, we next investigated the transcriptional regulation of CRISPLD2 in hBMSCs and found that transcription factor AP-2 alpha (AP2α), homeobox D9 (HOXD9), and transcription factor 4 (TCF4) are transcriptional repressors to modulate CRISPLD2 expression and osteogenic differentiation of hBMSCs. Moreover, AP2α regulates hBMSC osteogenic differentiation through 1) regulation of CRISPLD2 by directly binding to its transcription regulatory region (TRR) and 2) PI3K-AKT signaling pathway. In addition, we revealed a pro-osteogenic transcription factor, signal transducer and activator of transcription 4 (STAT4), whose expression was progressively decreased during in vitro expansion of hBMSCs. Knockdown of STAT4 in early passage hBMSCs significantly inhibited the cells’ osteogenic differentiation potential. Moreover, AAV-mediated STAT4 overexpression could retard the loss of osteogenic potential during in vitro expansion of hBMSCs. Our findings shed light on the molecular mechanisms of loss of osteogenic differentiation during hMSC expansion and provide potential therapeutic targets for hMSCs-based bone regeneration.

Date

4-3-2024

Committee Chair

Yao, shaomian

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Available for download on Wednesday, April 02, 2031

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