Degree

Doctor of Philosophy (PhD)

Department

Engineering Science

Document Type

Dissertation

Abstract

The research in this thesis is divided into two areas. 1) Studying the effects of cryopreservation and developing freezing protocols suitable for adipose derived stem cells and 2) Identifying the underlying mechanisms involved in the differentiation of ASCs especially into osteogenesis and endothelial-genesis using next generation sequencing.

In the part one (Chapter 2 and 3), a novel protocol to freeze ASCs and the effects of long-term cryopreservation on ASCs were studied. Heat shocking ASCs at 43°C for 1 hour resulted in the temporal expression of HSPs particularly HSP70 and HSP32 to reach maximum expression at 3 hours post heat shock treatment. Cryopreservation of heat shocked ASCs at this time point with cryoprotectants such as PVP or using 1% DMSO (minimal amount) enhanced the cell viability by 10.6% and 18.8% respectively without loss of osteogenic and adipogenic differentiation potential in comparison to their non-heat shocked ASCs. Therefore, this protocol can be used to replace the conventional cryopreservation protocols to avoid the harmful genotypical changes in ASCs. The effects of cryopreservation on the long-term (>=10 years) frozen ASCs were evaluated and compared with the short-term (3-7 years) frozen and fresh ASCs. The osteogenic differentiation potential was partially hampered in the long-term cryopreserved group whereas the adipogenic potential remained unaffected by freezing as well as the length of the storage time. The post-thaw viability and surface marker expression of ASCs also remained intact after decade long freezing process in relation with fresh ASCs.

In part two (chapters 4 and 5), the mRNA-sequencing of ASCs undergoing osteogenesis and micro-RNA- sequencing of ASCs undergoing endothelial-genesis was performed. The differential gene expression of ASCs undergoing osteogenesis on day 21 was determined using RNA-seq. Gene ontology functional analysis on the differentially expressed genes revealed the enrichment of ECM and angiogenesis along with ERK1/2 and JNK pathways. The expression of matrisome genes and ECM remodeling enzymes during osteogenesis has been elucidated. In addition, the expression of several pro-angiogenic ELR+ chemokines indicate the possible role of differentiating ASCs in the development of vasculature to accomplish functional bone formation. The expression of regulatory genes such as CTNNB1, TGBR2, JUN, FOS, GLI3, and MAPK3 suggest the regulation of osteogenesis through interplay between the WNT, TGF-β, JNK, HedgeHog and ERK1/2 pathways. Furthermore, the validation of RNA-Seq data was performed by QPCR that indicated the matching between the two. miRNAs sequencing was performed on the CD34+ ASCs during the period of endothelial differentiation by chemical media. Using the bioinformatics analysis, several miRNAs (both upregulated and downregulated) that could be potential candidates to induce endothelial-genesis in ASCs were determined. Based on the validated miRNA targets and their kegg analysis, 6 downregulated miRNAs with anti-angiogenic properties can be considered as potential candidates to induce endothelial-genesis. To upregulate the miRNAs, the miRNA-mimics were transfected into CD34+ ASCs and similarly for downregulation, the anti-miRNAs were used. Based on the VWF antibody staining and the expression of endothelial genes (VWF, VEGF, and CD31), miR-335-5p (400nM), miR-330-5p (800nM), miR-181a-5p (400nM, 800nM, and 1600nM), anti-miR-125a-5p (1600nM and 3200nM), anti-miR-155-5p (1600nM), and anti-miR-145-5p (3200nM) were selected as the potential miRNAs to induce endothelial-genesis of ASCs.

Date

11-20-2019

Committee Chair

Devireddy, Ram

DOI

10.31390/gradschool_dissertations.5118

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Available for download on Wednesday, October 28, 2026

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