StemSpan-XF, a specialized culture medium, plays a crucial role in expanding hematopoietic stem and progenitor cells (HSPCs) for research and clinical applications. This article delves into a study examining the colony-forming potential of CD34+ cells cultured in StemSpan-XF, specifically focusing on the variations observed across different CD34 expression levels.
This study utilized purified cord blood (CB)-derived CD34+ cells cultured in StemSpan-XF supplemented with StemSpan CD34+ Expansion Supplement and UM171 for seven days. Post-culture, cells were stained with anti-CD34 antibody and 7-AAD for sorting, further categorized into CD34bright, CD34med, and CD34- populations based on CD34 expression. Colony-forming unit (CFU) assays employing MethoCult H4034 Optimum were conducted to assess the colony-forming potential of each sorted population.
Both bulk-sorted and single cell-sorted populations were analyzed. Bulk-sorted cells were plated at a density of 400 cells/mL, while single cells were index-sorted into individual wells pre-filled with MethoCult. Following a 14-day incubation period, colonies were enumerated either manually or using STEMvision.
Results revealed a significant correlation between CD34 expression and colony-forming potential. The culture-expanded CD34bright cells exhibited the highest CFU frequency, approximately 50%, compared to around 20% for CD34med cells and 10% for CD34- cells. Furthermore, CD34bright cells generated a greater diversity of colony types, including more primitive colonies like CFU-GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte) and BFU-E (burst-forming unit-erythroid), which were scarcely observed in CD34med and CD34- populations.
Phenotypic analysis indicated that CFU-GEMM and a majority of BFU-E colonies originated from the CD34brightCD90+CD45RA- cell fraction, enriched for long-term hematopoietic stem cells (LT-HSCs). A smaller portion of BFU-E colonies arose from the CD34brightCD90-CD45RA- fraction, associated with short-term hematopoietic stem cells (ST-HSCs). Conversely, CD34brightCD90+/-CD45RA+ cells primarily yielded small CFU-G (granulocyte) and CFU-M (monocyte) colonies, consistent with their progenitor cell characteristics.
In conclusion, this study demonstrates that culture expansion in StemSpan-XF maintains the colony-forming potential of CD34+ cells, with CD34bright cells exhibiting superior CFU frequency and generating more diverse and primitive colony types. This underscores the importance of StemSpan-XF in supporting the ex vivo expansion of HSPCs for various applications. Comparable results are anticipated when using UM729 at a final concentration of 1µM in place of UM171.
