Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Brauchart, T.
Engineering of mesenchymal stromal cells to improve culture conditions for primary leukemia
Humanmedizin; [ Diplomarbeit ] Medizinische Universität Graz; 2024. pp. 67
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Reinisch Andreas
- Sill Heinz
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- Abstract:
- Introduction The bone marrow niche provides a distinctive microenvironmental area, with its great variety of cell types. Harboring the hematopoietic stem cells (HSC), this niche provides crucial elements and structures for their proliferation and differentiation and therefore, is elemental for hematopoiesis. A second stem cell, the mesenchymal stem cell (MSC), is also located in the bone marrow niche. The MSC is not only the progenitor of multiple different cell types, but also plays a necessary role in the proliferation of the HSC. Ex vivo attempts to expand HSCs, led to disappointing results, due to their high demands of cytokines and cell-cell interaction. Co-culture experiments tried to overcome this obstacle, but still relied on external cytokine substitution. Methods In this thesis we engineered human stromal cell lines (MSC-hTERT and HS27a), to overexpress human cytokines FLT3L, SCF, TPO and IL-6. Complementary DNA (cDNA) constructs together with a fluorescent marker (FLT3L_SCF_BFP and TPO_IL-6_BFP) were precisely introduced into safe harbor loci (HBB or AAVS1) by CRISPR/Cas9-mediated homology-directed repair (HDR) employing recombinant adeno-associated virus (rAAVs) as vectors for DNA template delivery. Precise knock–in was confirmed via an in-out PCR and cytokine levels were measured by an ELISA. Thereafter, human cytokine expressing stromal cells lines were co-cultured with either cytokine-dependent acute myeloid leukemia (AML) cell lines or cord blood-derived HSCs. Cell Expansion of AML cell lines and HSCs was quantified using flow cytometry. Additionally, HSPC subpopulation and colony-forming potential were evaluated. Results We successfully engineered a total of eight single cell-derived clones (four for TPO_IL-6_BFP, one for FLT3L_SCF_BFP and three with an introduction of both constructs) for the MSC-hTERT and 14 single cell-derived clones for the HS27a cell line (six for TPO_IL-6_BFP, five for FLT3L_SCF_BFP and four with an introduction of both constructs). Out of them four MSC-hTERT clones and nine HS27a clones provided measurable production of the introduced cytokines (p< 0.05). Co-culture with cytokine-expressing cell resulted in significant expansion of leukemia cells lines when compared to non-engineered wild type cells (MSC-KI:MSC-WT: p<0.001) or mono-culture conditions (MSC-KI: neg. ctrl: p<0.001) Interestingly, also non-engineered wild type stromal cells supported cell growth, when compared to mono-culture conditions (MSC-WT:neg. ctrl.: p<0.001). HSPC expansion, evaluated by total CD34+ cell counts, was significantly increased in co-cultures with cytokines expressing stromal cells samples (MSC-KI:neg. ctrl: p<0.001), while the percentage of CD34+ cells (p=0.051) and the total number of colonies was not significantly influenced by the secreted cytokines. Conclusion By utilizing the CRISPR/Cas9 system to precisely engineer a cytokine-expressing stromal cell, this study aimed to address the long existing challenges of culturing AML and HSPCs in vitro. The successful introduction of expression construct into MSCs resulted in the sufficient production of the human cytokines FLT3L, SCF, TPO, and IL-6. Co-culture experiments utilizing knocked-in MSCs further let to a substantial expansion and increased survival, supporting the theory of MSCs playing a crucial role in the bone marrow niche and highlight the critical role of these essential cytokines. While acknowledging certain limitations, including the non-physiological introduction of cytokines, high sample heterogeneity and small sample size, our research underlines the importance of MSCs in HSC maintenance and proliferation. The cytokine introduction via CRISPR/Cas9 holds promise for future advancements in culture settings, urging further studies to investigate the molecular mechanisms underlying MSC-HSC interactions and providing insights for long-term HSC culture possibilities.