Jarczak Justyna, Kieszek Patrycja, Ratajczak Mariusz Z, Kucia Magdalena
Laboratory of Regenerative Medicine, Preclinical Research and Technology Center, Medical University of Warsaw, Warsaw, Poland.
Stem Cell Institute at Graham Brown Cancer Center, University of Louisville, Louisville, CO, United States.
Front Cell Dev Biol. 2025 May 15;13:1590889. doi: 10.3389/fcell.2025.1590889. eCollection 2025.
Human hematopoietic stem/progenitor cells (HSPCs) are enriched in umbilical cord blood (UCB) among cell populations that express CD34 and CD133 (PROM1) antigens. These cells can be purified further and sorted by FACS as CD34LinCD45 and CD133LinCD45 cells. It has been postulated that the population of CD133 HSPCs is enriched for more primitive stem cells. To address this issue at the molecular level, we performed single-cell RNA-sequencing (scRNA-seq) and analyzed the transcriptome of both cell types. We optimized the available protocols of scRNA-seq of HSPC and described our laboratory experiences with the limited number of cells obtained from human UCB.
Herein, we report the results of scRNA-seq analysis paying special attention to the quality parameters of single cell libraries. We also present the similarities and differences in transcriptome between these cells (CD34Lin-CD45 and CD133Lin-CD45 HSPCs) and their subpopulations identified and visualized as clusters using uniform manifold approximation and projection (UMAP), stressing the need for an integrated analysis of both datasets, which may be merged and treated as "pseudobulk." We revealed that both populations do not differ significantly in gene expression, as evidenced by the very strong positive linear relationship between these cells (R = 0.99).
To obtain solid results that allow to draw conclusions that would have a biological translation, all parts of the scRNA-seq experiment are crucial and must be carried out with due care: cell sorting, single cell libraries preparation, quality control, and data analysis. The idea of working with sorted material instead of the typical use of a full pellet of blood cells was right and confirmed the possibility of HSPC analysis, even with a limited number of cells.
在表达CD34和CD133（PROM1）抗原的细胞群体中，人造血干/祖细胞（HSPCs）在脐带血（UCB）中富集。这些细胞可以进一步纯化，并通过荧光激活细胞分选术（FACS）分选成CD34LinCD45和CD133LinCD45细胞。据推测，CD133 HSPCs群体富含更原始的干细胞。为了在分子水平上解决这个问题，我们进行了单细胞RNA测序（scRNA-seq），并分析了这两种细胞类型的转录组。我们优化了现有的HSPCs的scRNA-seq方案，并描述了我们实验室处理从人脐带血中获得的有限数量细胞的经验。
在此，我们报告scRNA-seq分析的结果，特别关注单细胞文库的质量参数。我们还展示了这些细胞（CD34Lin-CD45和CD133Lin-CD45 HSPCs）及其使用均匀流形近似和投影（UMAP）识别并可视化为聚类的亚群之间转录组的异同，强调需要对这两个数据集进行综合分析，这两个数据集可以合并并当作“伪批量”处理。我们发现这两个群体在基因表达上没有显著差异，这些细胞之间非常强的正线性关系证明了这一点（R = 0.99）。
为了获得可靠的结果，以便得出具有生物学转化意义的结论，scRNA-seq实验的所有部分都至关重要，必须谨慎进行：细胞分选、单细胞文库制备、质量控制和数据分析。使用分选后的材料而不是典型的完整血细胞沉淀进行研究的想法是正确的，并证实了即使细胞数量有限，HSPC分析也是可行的。
