Department of Medicine, Division of Hematology, Washington University School of Medicine, St. Louis, MO, United States.
Department of Medicine, Division of Hematology, Washington University School of Medicine, St. Louis, MO, United States; Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, United States; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai School of Medicine, New York, NY, United States.
Stem Cell Res. 2022 Jul;62:102808. doi: 10.1016/j.scr.2022.102808. Epub 2022 May 8.
To achieve efficient, reproducible differentiation of human pluripotent stem cells (hPSCs) towards specific hematopoietic cell-types, a comprehensive understanding of the necessary cell signaling and developmental trajectories involved is required. Previous studies have identified the mesodermal progenitors of extra-embryonic-like and intra-embryonic-like hemogenic endothelium (HE), via stage-specific WNT and ACTIVIN/NODAL, with GYPA/GYPB (CD235a/b) expression serving as a positive selection marker for mesoderm harboring exclusively extra-embryonic-like hemogenic potential. However, a positive mesodermal cell-surface marker with exclusively intra-embryonic-like hemogenic potential has not been identified. Recently, we reported that early mesodermal expression of CDX4 critically regulates definitive HE specification, suggesting that CDX4 may act in a cell-autonomous manner during hematopoietic development. To identify CDX4+ mesoderm, we performed single cell (sc)RNAseq on hPSC-derived mesodermal cultures, revealing CDX4 expressing mesodermal populations were uniquely enriched in the non-classical MHC-Class-1 receptor CD1D. Flow cytometry demonstrated approximately 60% of KDR+CD34-CD235a- mesoderm was CD1d+, and CDX4 was robustly enriched within CD1d+ mesoderm. Critically, only CD1d+ mesoderm harbored CD34+ HOXA+ HE with multilineage erythroid-myeloid-lymphoid potential. Thus, CDX4+CD1d+ expression within early mesoderm demarcates an early progenitor of HE. These insights may be used for further study of human hematopoietic development and improve hematopoietic differentiation conditions for regenerative medicine applications.
为了实现人类多能干细胞（hPSC）向特定造血细胞类型的高效、可重复分化，需要全面了解涉及的必要细胞信号和发育轨迹。以前的研究已经通过阶段特异性的 WNT 和 ACTIVIN/NODAL 确定了胚外样和胚内样造血内皮（HE）的中胚层祖细胞，GYPAB（CD235a/b）的表达作为仅具有胚外样造血潜能的中胚层的阳性选择标记。然而，具有仅胚内样造血潜能的阳性中胚层细胞表面标记尚未被鉴定。最近，我们报道早期中胚层表达的 CDX4 对确定的 HE 特异性具有关键调节作用，这表明 CDX4 可能在造血发育过程中以细胞自主的方式发挥作用。为了鉴定 CDX4+中胚层，我们对 hPSC 衍生的中胚层培养物进行了单细胞（sc）RNAseq，结果表明 CDX4 表达的中胚层群体在非经典 MHC 类-1 受体 CD1D 中被独特地富集。流式细胞术表明，大约 60%的 KDR+CD34-CD235a-中胚层为 CD1d+，并且 CDX4 在 CD1d+中胚层中得到了强烈富集。关键的是，只有 CD1d+中胚层具有具有多谱系红系-髓系-淋巴系潜能的 CD34+HOXA+HE。因此，早期中胚层中的 CDX4+CD1d+表达标志着 HE 的早期祖细胞。这些见解可用于进一步研究人类造血发育，并改善再生医学应用中的造血分化条件。
