The frequency of multipotent CD133(+)CD45RA(-)CD34(+) hematopoietic stem cells is not increased in fetal liver compared with adult stem cell sources

Abstract

The cell surface marker CD133 has been used to describe a revised model of adult human hematopoiesis, with hematopoietic stem cells and multipotent progenitors (HSCs/MPPs: CD133(+)CD45RA(-)CD34(+)) giving rise to lymphomyeloid-primed progenitors (LMPPs: CD133(+)CD45RA(+)CD34(+)) and erythromyeloid progenitors (EMPs: CD133(low)CD45RA(-)CD34(+)). Because adult and fetal hematopoietic stem and progenitor cells (HSPCs) differ in their gene expression profile, differentiation capabilities, and cell surface marker expression, we were interested in whether the reported segregation of lineage potentials in adult human hematopoiesis would also apply to human fetal liver. CD133 expression was easily detected in human fetal liver cells, and the defined hematopoietic subpopulations were similar to those found for adult HSPCs. Fetal HSPCs were enriched for EMPs and HSCs/MPPs, which were primed toward erythromyeloid differentiation. However, the frequency of multipotent CD133(+)CD45RA(-)CD34(+) HSPCs was much lower than previously reported and comparable to that of umbilical cord blood. We noted that engraftment in NSG (NOD scid gamma [NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ]) mice was driven mostly by LMPPs, confirming recent findings that repopulation in mice is not a unique feature of multipotent HSCs/MPPs. Thus, our data challenge the general assumption that human fetal liver contains a greater percentage of multipotent HSCs/MPPs than any adult HSC source, and the mouse model may have to be re-evaluated with respect to the type of readout it provides.

Figure 1. Phenotypical and functional in vitro characterization of FL-, UCB-, and PBSC- derived CD34⁺ subpopulations

(A) Revised model of human hematopoiesis (BM and UCB) with HSCs/MPPs giving rise to either lympho-myeloid or erythro-myeloid progenitor cells. HSPCs with erythroid differentiation potential are highlighted in red. Abbreviations: MPP: multipotent progenitor; LMPP: lympho-myeloid primed progenitor; EMP: erythro-myeloid progenitor; MLP: multilymphoid progenitor; GMP: granulocyte (neutrophil)-macrophage progenitor; EoBP: eosinophil-basophil progenitor; MEP: megakaryocyte-erythrocyte progenitor. (B) Phenotype of functionally distinct hematopoietic stem and progenitor cell subpopulations. (C, D and E) Flow cytometric analysis of MACS-purified FL-, UCB-, and PBSC-derived CD34⁺ cells after exclusion of dead cells/debris (FSC/SSC) and non-hematopoietic/endothelial CD45⁻ cells; subdivision of CD45⁺CD34⁺ cells into CD45⁺CD34⁺CD133⁺CD45RA⁻ HSC/MPP⁻, CD45⁺CD34⁺CD133⁺CD45RA⁺ LMPP⁻, CD45⁺CD34⁺CD133^lowCD45RA⁻ EMP⁻, and CD45⁺CD34⁺CD133^lowCD45RA⁺ GMP-enriched fractions. (F, G and H) Frequency of HSCs/MPPs, LMPPs, EMPs and GMPs within FL-, UCB-, and PBSC-derived CD34⁺ populations. (I, J and K) Frequency of erythroid (BFU-E: burst forming unit-erythrocyte), myeloid (CFU [colony-forming unit]-M [macrophage], -G [granulocyte] and -GM [granulocyte-macrophage]), and erythro-myeloid (CFU-MIX = granulocyte-erythrocyte-megakaryocyte-macrophage) colonies obtained from sort-purified FL, UCB and PBSC CD34-subpopulations. (L, M and N) Primary CFCs of HSC/MPP, LMPP, EMP, and GMP populations were harvested and replated. Secondary colony-forming potential represents total colony count without discrimination of colony-subtypes.

Figure 2. Phenotypical and functional characterization of culture-derived FL, UCB, and PBSC CD34⁺ subpopulations

(A–C) Sort-purified FL-, UCB-, and PBSC-derived HSC/MPP, LMPP, EMP, and GMP subpopulations (post sort, upper row) were cultured in StemSpan supplemented with SCF, TPO and Flt3-L (100ng/ml each). Phenotype, composition, and proliferation of arising progeny was analyzed on day 3 (post culture, lower row). (D–F) On day 3 of culture, HSC/MPP-derived CD133/CD45RA-subpopulations were sort-purified, introduced into colony-forming cell assay and ability to form different colony-subtypes quantified (all statistics mean ± SEM; *: p<0.05, **: p<0.01, ***: p<0.001).

Figure 3. Engraftment potential of FL-derived CD34⁺ cells

(A) Flow-cytometric quantification of human CD45⁺ (hCD45⁺) cells in the peripheral blood (PB) of transplanted mice 8–18 weeks post transplant. (B and C) Representative flow-cytometric analysis of human B cells (CD20⁺), monocytes (CD14⁺), T cells (CD3⁺/CD4⁺/CD8⁺/CD4⁺CD8⁺) and CD34⁺ progenitor cells in PB, thymus, spleen, and bone marrow (BM) of reconstituted mice (week 18). (D–G) Average frequency of human B cells, T cells, monocytes, and CD34⁺ cells within hCD45⁺ fractions (left axis) as well as T cell-subtypes within human CD3⁺ cell populations (right axis) (H) Representative flow-cytometric analysis and (I) frequency of the HSC/MPP-, LMPP-, EMP-, and GMP subpopulations in thymus-, spleen-, and BM-resident CD34⁺ fractions (week 18). (J) Colony-forming potential of sort-purified human HSC/MPP-, LMPP-, EMP-, and GMP-enriched subpopulations from murine BM. (K) Sort-purified human HSC/MPP- and LMPP-enriched subpopulations from murine BM samples (post sort) were cultured in StemSpan supplemented with SCF, TPO and Flt3-L (100ng/ml each) and composition/proliferation of arising progeny analyzed on day 3 post culture (all statistics mean ± SEM; *: p<0.05, **: p<0.01, ***: p<0.001).