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Hematopoietic stem and progenitor cell heterogeneity and susceptibility to HIV-1
Thesis (PhD (Medical Immunology))--University of Pretoria, 2019.
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| Format: | Thesis |
| Language: | English |
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University of Pretoria
2020
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| _version_ | 1867613459802226688 |
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| access_status_str | Open Access |
| author2 | Pepper, Michael Sean |
| author_browse | Pepper, Michael Sean |
| author_facet | Pepper, Michael Sean |
| collection | Thesis |
| dc_rights_str_mv | © 2019 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Thesis (PhD (Medical Immunology))--University of Pretoria, 2019. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/73434 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:36:29.578Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/73434 Hematopoietic stem and progenitor cell heterogeneity and susceptibility to HIV-1 Pepper, Michael Sean juanitamellet@yahoo.co.uk Mellet, Juanita UCTD Cellular and Molecular Biology SDG-03: Good health and well-being Thesis (PhD (Medical Immunology))--University of Pretoria, 2019. Umbilical cord blood (UCB) is a rich source of hematopoietic stem and progenitor cells (HSPCs). There are however limitations to using UCB as a regular source for hematopoietic stem cell transplantation (HSCT). The number of CD34+ HSPCs is limited, while a minimum number of CD34+ HSPCs is required for HSCT, which cannot always be achieved. New developments in HSCT are currently underway to expand current applications and improve safety and efficacy. This necessitates efficient ex vivo expansion of these cells to therapeutic numbers. HSCT is being investigated in therapies for non-hematopoietic disorders with the goal of replacing diseased cells or tissue with healthy cells. HSPC-based gene therapy strategies are becoming attractive applications of corrective ex vivo gene transfer given the reconstitutive potential of HSCT. The success of these strategies for the treatment of monogenic disorders resulted in the application of HSPC gene therapy being considered for other diseases such as the human immunodeficiency virus (HIV). The optimal isolation method was determined for increased HSPC purity and viability by testing two different methods, magnetic activated cell sorting (MACS) and fluorescent activated cell sorting (FACS). FACS was considered optimal for our purposes and was used to isolate CD34+ HSPCs for subsequent experiments. Different commercially available serum-free media were tested and compared to standard medium supplemented with foetal bovine serum (FBS). All commercial serum-free media outcompeted the standard medium based on viability and proliferation. Building on the previous work, StemSpan ACF was used to test combinations of cytokines for their expansion potential. The combination containing FLT3L, SCF, TPO, IL-3 and G-CSF resulted in the greatest expansion of HSPCs. The effect of StemRegenin-1 (SR1) on the expansion of HSPCs was explored by adding SR1 to the above-mentioned cytokine combinations. This resulted in minor effects on HSPC expansion based on viability and immunophenotype. Similarly, it resulted in only two significantly downregulated genes, cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) and erythrocyte membrane protein band 4.1-like 3 (EPB41L3), in both CD34+ and CD34– cells compared to non-treated controls. The use of CD34+ HSPCs exclusively expanded with SR1 would be beneficial in cases where the HSPC cell dose of the initial harvested cell therapy product is suboptimal and therefore not a feasible option for HSCT on its own. Single-cell RNA sequencing was performed on CD34+ HSPCs and four populations were identified, which is in line with previous publications. HSPC gene therapy is a promising approach to treat HIV. However, this type of approach would require the presence of significant numbers of long-term repopulating HSPCs to enable successful long-term engraftment of gene-modified cells. One aspect that could result in this approach not succeeding is the presence of proviral DNA in HSPCs. It would therefore be important to identify a population of HSPCs that is resistant to HIV infection. It was therefore investigated whether HSPCs from leukapheresis products are susceptible to infection with HIV and whether a subset of HSPCs exists that is resistant to infection to use in HIV gene therapies. Unfortunately, this could not be achieved due to loss of viability of HSPCs from leukapheresis products. Personal Funding: National Research Foundation (NRF), Poliomyelitis Research Foundation (PRF) and the Institute for Cellular and Molecular Medicine (ICMM). Research Funding: The South African Medical Research Council Extramural Unit for Stem Cell Research and Therapy and in terms of the SAMRC’s Flagship Award Project [SAMRC-RFA-UFSP-01-2013/STEM CELLS] as well as the Institute for Cellular and Molecular Medicine of the University of Pretoria. em2026 Immunology PhD (Medical Immunology) Unrestricted SDG-03: Good health and well-being 2020-02-19T14:05:30Z 2020-02-19T14:05:30Z 2020 2019 Thesis Mellet, J 2019, Hematopoietic stem and progenitor cell heterogeneity and susceptibility to HIV-1, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd http://hdl.handle.net/2263/73434 A2020 http://hdl.handle.net/2263/73434 en © 2019 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | UCTD Cellular and Molecular Biology SDG-03: Good health and well-being Hematopoietic stem and progenitor cell heterogeneity and susceptibility to HIV-1 |
| title | Hematopoietic stem and progenitor cell heterogeneity and susceptibility to HIV-1 |
| title_full | Hematopoietic stem and progenitor cell heterogeneity and susceptibility to HIV-1 |
| title_fullStr | Hematopoietic stem and progenitor cell heterogeneity and susceptibility to HIV-1 |
| title_full_unstemmed | Hematopoietic stem and progenitor cell heterogeneity and susceptibility to HIV-1 |
| title_short | Hematopoietic stem and progenitor cell heterogeneity and susceptibility to HIV-1 |
| title_sort | hematopoietic stem and progenitor cell heterogeneity and susceptibility to hiv 1 |
| topic | UCTD Cellular and Molecular Biology SDG-03: Good health and well-being |
| url | http://hdl.handle.net/2263/73434 |