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HIV-exposed uninfected umbilical cord blood haematopoietic stem/progenitor cells differ immunophenotypically from those from HIV unexposed umbilical cord blood but have similar expansion and colony-forming properties in vitro

Bone Marrow Transplantation volume 60, pages 897–901 (2025)Cite this article

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South Africa (SA) is a human immunodeficiency virus (HIV) endemic country in which allogeneic haematopoietic stem cell transplantation (HSCT) is performed at a low rate of approximately 2,5/million population per annum. A deficit in donors constitutes a major obstacle, and efforts advocating for an umbilical cord blood (UCB) bank [1], while concurrently improving donor numbers from African ethnolinguistic groups, are underway. Although haploidentical HSCT is gaining favour globally, UCB has particular advantages in ethnically diverse populations [2] due to human leukocyte antigen (HLA) typing being at ≥4/6 or ≥4/8 loci [3] instead of 8/8-10/10; ex vivo expansion of UCB haematopoietic stem/progenitor cells (HSPCs) is an additional advantage [4]. In South Africa, the prevalence of pregnant women living with HIV (LWH) is reported to be 30-40% [5]. However, <2% of these mothers will give birth to HIV-infected infants [5] as a consequence of successful preventive antiretroviral (ARV) strategies. Haematological abnormalities have been reported in HIV exposed uninfected (HEU) children, with zidovudine (AZT) being implicated [6]. We have recently advocated for the inclusion of HIV exposed or infected donors in the repertoire of donors for HSCT in South Africa, given that it is an HIV endemic country [7]. In addition to adult donors LWH, we proposed that HEU UCB should be considered once sufficient evidence for the optimal functioning of these HSPCs had been obtained. The first step in determining the potential of using HEU units for HSCT was to perform extensive in vitro analysis as a basis for future in vivo analysis. This study thus provides information on the characteristics of HEU HSPCs by determining their expansion capacity, in the event that HSPC expansion is considered prior to infusion in HSCT in the future. Immunophenotypic analysis of cells expanded with StemRegenin 1 (StemCell™ Technologies, Canada, at a final in-well concentration of 1 µM) [8] was then compared to a vehicle control (VC; 0.01% DMSO and cytokines only) to determine if expansion changes the potential Lin-CD34+ sub-groups in HIV exposed HSPCs. Finally, gene expression and colony-forming ability in vitro, were assessed to determine if potential gene pathways or colony formation are affected by HIV exposure. We collected UCB from HIV negative and HIV exposed infants post elective caesarean section at term. All samples tested negative for HIV-1 by GeneXpert analysis using HIV-1 Qual XC cartridges (Cepheid, USA). For HEU samples, mothers undergoing elective caesarean section were on ARVs and were virologically suppressed (at least one viral load [VL] <100 copies/mL within 3 months prior to delivery), and infant HIV polymerase chain reaction (PCR) (within the first few days of birth) was negative (Fig. S1D). Eleven mothers living with HIV consented to UCB collection with five samples meeting the above inclusion criteria. Of these, 4 had sufficient cell numbers to allow for a complete set of experiments. All mothers were on fixed drug combination ARVs, four on TLD (Tenofovir disoproxil fumarate [TDF], Lamivudine, Dolutegravir) and one on TEE (TDF, Emtricitabine, Efavirenz) (Fig. S1D).

Mononuclear cells (MNCs) were isolated using a CD34+ magnetic kit (Miltenyi, Germany) (>95% purity obtained, Fig. S1A) and plated at 1 × 104 cells/well in a 24-well plate in triplicate in Stemspan animal origin free medium (StemCell™ Technologies, Canada) for each condition (StemRegenin 1 [SR1] and vehicle control [control]). Recombinant human growth factors were added, each at 100 ng/mL: Stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3L), granulocyte colony-stimulating-factor (G-CSF) and interleukin-3 (IL-3) (all from Life Technologies, Thermo Fisher Scientific, USA). Cells were cultured for 7 days in 5% CO2 at 37 °C. Between 5 × 105-1 × 106 MNCs were stained for immunophenotypic analysis using Lin-FITC (clones:CD3:UCHT1; CD14:HCD14; CD16:3G8; CD19:HIB19; CD20:2H7; CD56:HCD56), CD117-PE (clone:104D2), CD90-BV510 (clone:5E10) and CD49f-SB780 (clone:G0H3) from BioLegend, San Diego, USA; CD34-APC AF700 (clone:581), CD38-ECD (clone:LS198-4-3) and CD45RA-APC (clone:2H4) from Beckman Coulter, Miami, USA; and CD133-PE-Violet 770 (clone:REA753) from Miltenyi, Germany. Data was acquired on a CytoFLEX flow cytometer (Beckman Coulter, Miami, USA). Compensated, logicle-transformed, pre-gated data (see Fig. S1B for D0 and Fig. S1C for D7 gating strategy) was exported to the Cytobank platform using the Kaluza Cytobank plugin (http://www.cytobank.org; Beckman Coulter, Miami, USA). After dimensionality reduction, the FlowSOM clustering algorithm (self-organizing map) was applied. Statistically significant differences (p-value < 0.05) in FlowSOM metaclusters (MCs) were identified using the Kruskal Wallis test in the statistical inference tool in the Cytobank platform. For CFUs, 200 cells in 4 µL were added in 500 µL Methocult medium per well (StemCell™ Technologies, Canada) in three wells of a scored 24-well plate. Colonies were identified on Day 14 (D14) using a Zeiss Axio-microscope with an Axiocam 1Cc-5 (Zeiss, Germany) and a CFU atlas as a guide (R&D systems, USA). Staining was then performed using CD235a-PE (clone:11E4B-7-6), CD71-APC-AF750 (clone:YDJ1.2.2), CD41-ECD (clone:P2), CD15-KO (clone:80H5), CD14-APC-AF700 (clone:RM052), CD19-PC5.5 (clone:J3-119), CD20-PC5.5 (clone:B9E9), CD3-PC5.5 (clone:UCHt1), CD56-PC5.5 (clone:NP01) and CD33-APC (clone:D3HL60.251) [All antibodies, Beckman Coulter, Miami, USA]. A second tube was stained with 7-AAD (Beckman Coulter, Miami, USA).

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Fig. 1: Comparison between HEU and HUU samples in HSPC number after 7 days in culture, and colony forming units after 14 days in culture.
Fig. 2: Differences in immunophenotype and gene expression between HEU and HUU samples.

Data availability

Microarray data are available at GEO under accession number GSE253897. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE253897For original flow cytometry data files, please contact michael.pepper@up.ac.za.

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Funding

This research was supported by the South African Medical Research Council (SAMRC) Extramural Unit for Stem Cell Research and Therapy, and the University of Pretoria through the Institute for Cellular and Molecular Medicine (to MSP).

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Authors and Affiliations

  1. Institute for Cellular and Molecular Medicine, Department of Immunology, Faculty of Health Sciences, University of Pretoria, South Africa; and SAMRC Extramural Unit for Stem Cell Research and Therapy, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa

    Candice Laverne Hendricks, Juanita Mellet & Michael Sean Pepper

  2. Beckman Coulter Life Sciences, Pretoria, South Africa

    Voula Stivaktas

  3. Department of Oral and Maxillofacial Pathology, School of Dentistry, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa

    Melvin Ambele

Authors
  1. Candice Laverne Hendricks
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  2. Juanita Mellet
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  3. Voula Stivaktas
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  4. Melvin Ambele
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  5. Michael Sean Pepper
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Contributions

Conceptualisation: CLH, JM, MSP; Methodology: CLH, JM, MSP; Investigation: CLH, JM; Writing- Original draft: CLH; Writing- Review and Editing: CLH, JM, VS, MA, MSP; Formal analysis: CLH, VS, MA; Data curation: CLH, VS, MA; Resources and funding acquisition: MSP; Supervision: JM and MSP.

Corresponding author

Correspondence to Michael Sean Pepper.

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Competing interests

MSP has received research grants from the South African Medical Research Council, the Bill and Melinda Gates Foundation, the South African National Health Laboratory Service Research Trust, The National Research Foundation (South Africa) and the Wellcome Trust. MSP is a co-founder and non-remunerated board member of Antion Biosciences and Altera Biosciences and is also CSO of the latter. CLH is a non-remunerated board member of DKMS Africa, a not-for-profit organisation.

Ethics approval

Ethics approval for this work was obtained from the University of Pretoria Faculty of Health Sciences Research Ethics Committee, approval number 102/2020. All enrolled mothers gave written informed consent, for collection of umbilical cord blood after delivery of the infant, for the purposes of the study. All methods were performed in accordance with the relevant guidelines and regulations.

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Hendricks, C.L., Mellet, J., Stivaktas, V. et al. HIV-exposed uninfected umbilical cord blood haematopoietic stem/progenitor cells differ immunophenotypically from those from HIV unexposed umbilical cord blood but have similar expansion and colony-forming properties in vitro. Bone Marrow Transplant 60, 897–901 (2025). https://doi.org/10.1038/s41409-025-02574-6

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