Phenotype and Function of Human Gut Hematopoietic Stem Cells and Progenitors

Abstract

Lin−CD45+CD34+ cells have been reported in human small intestine and liver, but further phenotypic or functional analysis was not described[1][2]. We utilized flow cytometry and cytometry by time of flight (CyTOF) to study the composition and phenotype of human gut hematopoietic stem cells (HSCs) and hematopoietic progenitors (HPs) from deceased donors. Colony-forming-cell (CFC) assays, long-term-culture-initiating-cell (LTC-IC) assays and humanized mouse models were established to further evaluate the differentiation potential of gut HSCs/HPs. HSCs and multiple types of HPs, including multipotent progenitors (MPP), lymphoid-primed multipotent progenitors (LMPP), common lymphoid progenitors (CLP), myeloid progenitors (MP), and unclassified progenitors co-expressing CD56, were detected in human intestinal mucosa and mesenteric lymph nodes (MLNs). Ileum lamina propria lymphocytes (LPL) included significantly higher percentages of HSCs than intraepithelial lymphocytes (IEL). Both IEL and LPL showed significantly lower percentages of CLP compared to MLNs. From jejunum or ileum specimens of ITx patients collected from their stoma revision/closure (POD47- 1544), variable levels of Lin−CD45+CD34+ cells were detected (Figure 1A). However, when gating on HSCs, donor cells (>90%) predominated over recipient cells in four patients with chimerism, regardless of their ages or early clinical outcomes (Figure 1B-C). Lin−CD45+CD34+ cells from ileum LP and bone marrow (BM) demonstrated similar properties in viSNE plots from CyTOF analysis (Figure 2), suggesting similar functional phenotypes. Both ileum LP and BM Lin−CD45+CD34+ cells contained subpopulations expressing the thymus-homing marker CCR9, and T/NK lineage polarized progenitors coexpressing CD45RA and CD7. Additionally, CFC assays showed that both ileum LP and BM Lin−CD34-enriched cells can form myeloid/erythroid colonies, although ileum LP CD34 cells were less efficient than BM CD34 cells. LTC-IC assay using single cell sorted ileum LP HSC co-culture with murine feeder cells suggested myeloid/lymphoid differentiation potential of human gut HSCs. Experiments using human gut Lin−CD45+CD34+ cells to reconstitute humanized mice are ongoing.FIGURE 1: (A) Lin−CD45+CD34+ cells were detected in human intestinal mucosa long-term post-Tx (POD47-1544) (B) Donor-derived hematopoietic stemcells (HSCs) and/or multiple types of hematopoietic progenitors (HPs)were detected in transplanted small bowel specimens. (C) Composition of HSCs/HPs when gating on donor (upper panel) or recipient (lower panel) Lin−CD45+CD34+ cells.FIGURE 2: Lin−CD45+CD34+ cells from bone marrow (BM) and ileum lamina propria (LP) located in the same region of the viSNE plot from CyTOF analysis of individual organ donor, suggesting the similarity of their phenotype and origin.Taken together, our data suggest that human gut HSCs/HPs may be functionally similar to BM HSCs/HPs that have thymus-homing ability and can give rise to multiple lineages, which likely contribute to the long-persistent mixed chimerism post-ITx. Research reported in this publication was performed in the Columbia Center for Translational Immunology (CCTI) Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. CyTOF study was performed in collaboration with the Human Immune Monitoring Core (HIMC) of Icahn School of Medicine at Mount Sinai.