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Developmental Hierarchy and Lineage Commitment in Hematopoietic Progenitors

(Lai AY and Kondo M, J Exp Med, 2006:203;8:1867) 
Summarized by Robert Margolin and Yusra Abidi 

Figure 1

Figure 2

Lay Summary 

Lineage commitment and differentiation of Hematopoietic Stem Cells (HSCs) is a poorly understood and highly debated topic in the field of stem cell biology.  The study conducted by Lai et al. challenged the classic model of HSC differentiation (Figure 1), or the formation of white blood cells, red blood cells, and platelets from a hematopoietic stem cell.  The study postulated an alternative pathway for HSC differentiation (Figure 2).  Multipotent progenitors (MPPs) – cells slightly more mature than a stem cell that are still capable of forming all blood cells – were derived from 6-8 week-old mice and characterized into three distinct subpopulations based on expressions of cell surface markers Flt3 and vascular cell adhesion molecule 1 (VCAM-1) in both the laboratory and mice.  Flt3 and VCAM-1 are both proteins expressed on the surface of cells and used in the study as cell “i.d. tags.”  The cells were then were tested for expression of several lymphoid (white blood cells) and myeloid (red blood cells, platelets) related surface markers. 

HSCs and MPPs were separated by Fluorescence-activated cell sorter (FACS). This method separated cells based on expression of surface markers.  The three cells separated were long term HSCs, short term HSCs and MMPs, which all have different cell surface markers. Long term HSCs are less mature cells and have greater self renewal ability than short term HSCs followed by MPPs which loose the ability to self renew and are most mature.  MPPs were then subdivided into three different populations: Flt3(low)VCAM-1+, flt3(high)VCAM-1+, and Flt 3(high)VCAM-1-.  

Myeloid differentiation potential of MPPs was studied under laboratory conditions; results showed that only Flt 3(low)VCAM-1+ MPPs produced adequate red blood cell colonies, and mixed colonies of other myeloid cells.  Furthermore, only Flt3(low)VCAM-1+ MPPs displayed adequate levels of GATA1 and EpoR, gene expression that is essential in the formation of red blood cells.  The authors also studied red blood cell differentiation potential of MPPs in mice, and found that Flt3(low)VCAM-1+ MPPs  contributed to the greatest amount of platelets derived from donor cells, indicating their red blood cell differentiation potential. 

Results further showed that while all MPPs produce B and T cells in mice, lymphocyte production by Flt3(low)VCAM-1+ was much slower than the other two MPPs, suggesting that Flt3(low)VCAM-1+ MPPs are the most primitive type of MPP with complete potential to produce multiple lineages.  These results indicate a hierarchy of MPPs with the Flt3(low)VCAM-1+ MPPs at the top, followed by the more mature Flt3(high)VCAM-1+ MPPs, which finally differentiate into Flt3(high)VCAM-1- MPPs. 

Next, lymphoid lineage priming, or lymphoid commitment in MPPs was studied via determining which MPPs express the lymphoid lineage associated genes, Rag1 and IL-7Rα.  Results indicated that RAG 1 and IL-7Rα were absent in Flt3(low)VCAM-1+ and Flt3(high)VCAM-1+ MPPs, but upregulated in Flt3(high)VCAM-1- MPPs.  Thus, Lymphoid-related genes are primed in Flt3(high)VCAM-1- MPPs, causing them to differentiate into white blood cells. 
 

Scientific Summary 

      Lineage commitment and differentiation of hematopoietic Stem Cells (HSCs) is a poorly understood and highly debated topic in stem cell discourse.  The classic model of hematopoietic differentiation holds that lineage commitment occurs when a multipotent progenitor (MPP) commits to either the myeloid or lymphoid lineages (Figure 1).  The study conducted by Lai et al. challenged the classic model of HSC differentiation and postulated an alternative pathway for HSC lineage commitment (Figure 2).  Lai et al. characterized multipotent progenitors (MPPs) into three distinct subpopulations based on expressions of cell surface markers Flt3 and vascular cell adhesion molecule 1 (VCAM-1) in vitro and in vivo.  In vivo studies were conducted on 6-8 week, and 8-12 week-old C57BL/Ka-Thy-1.1 and C57BL/Ka-Thy-1.1-Ly5.2 (CD45.1) wild type mice. The authors emphasize asymmetric lineage divergence of lymphoid and myeloid progenitors from a common multipotent HSC. 

      HSCs and MPPs were separated by Fluorescence-activated cell sorter (FACS) and analyzed based on expression of surface antigens Thy-1.1 and Flt3 (figure 1A).  Long term HSCs populations were abundant in the Thy-1.1(low) flt3- bone marrow fraction, while short term HSCs were found in the Thy-1.1(low) Flt3(low) fraction.  MPPs were found in the Thy-1.1- Flt3+ bone marrow fraction.  MPPs were then subdivided into three different populations using FACS based on their expression of VCAM-1 and flt3 markers after two rounds of sorting (figure 1B).  The three distinct sets of MPPs include populations that were Flt3(low)VCAM-1+, flt3(high)VCAM-1+, and Flt 3(high)    VCAM-1-. 

      Previous research discrepancies over MPP and CMP differentiation and erythroid lineage divergence led the authors of this study to speculate the differentiation potential of VCAM-1+ MPPs more closely.  To study the megakaryocyte/erythroid (MegE) differentiation potential of MPPs in vitro, Lai et al. compared colony formation by MPPs cultured in methylcellulose, stem cell factor, IL-3, IL-6, erythropoietin, and thrombopoietin.  The results indicated that only Flt 3(low) VCAM-1+ MPPs produced adequate erythroid (CFU-E) or mixed (CFU-GEMM) colonies (figure 2A).  Furthermore, only Flt3(low)VCAM-1+ MPPs expressed adequate levels of GATA1 and EpoR – genes essential for erythropoiesis.  To study MegE differentiation potential of MPPs in vivo, purified MPPs from enhanced green fluorescence protein (EGFP) mice were injected into lethally irradiated host mice.  Peripheral blood from reconstituted mice was collected and analyzed using FACS two weeks after injection to detect platelets derived from the donor mice. The results showed that Flt3(low)VCAM-1+ MPPs  contributed to the greatest amount of platelet chimerism, indicating their erythroid differentiation potential. 

      To analyze the lymphoid lineage differentiation potential of MPPs, lai et al. collected peripheral blood from reconstituted mice and used FACS to analyze it for donor derived B (220+) and T (CD3+) cells 3-5 weeks after injection.  Results showed that while all MPPs produce B and T cells in vivo, lymphocyte production by Flt3(low)VCAM-1+ was much slower than the other two MPPs, suggesting that Flt3(low)VCAM-1+ MPPs are the most primitive type of MPP with complete multilineage differentiation potential.  These results indicate a hierarchy of MPPs with the Flt3(low)VCAM-1+ MPPs at the top, followed by the more mature Flt3(high)VCAM-1+ MPPs, and finally Flt3(high)VCAM-1- MPPs. 

      To evaluate lymphoid lineage priming in MPPs, the authors used quantitative PCR to analyze lymphoid lineage associated genes, Rag1 and IL-7Rα expression in the three subsets of MPPs.  Results indicated that RAG 1 and IL-7Rα were absent in Flt3(low)VCAM-1+ and Flt3(high)VCAM-1+ MPPs, but upregulated in Flt3(high)VCAM-1- MPPs.  These results indicate that initiation of lymphoid lineage priming and loss of erythroid differentiation ability are not synchronous.  The authors of this study postulate that the loss of MegE differentiation ability occurs first, with Flt3(high)VCAM-1+ MPPs retaining GM differentiation ability, possibly without activation of the lymphoid lineage.  Lymphoid-related genes are primed in Flt3(high)VCAM-1- MPPs in vivo, causing them to differentiate into lymphocytes. 

      To analyze the ability of MPPs to differentiate into CMPs in vivo, Lai et al. injected Flt3(low)VCAM-1+ and Flt3(high)VCAM-1+ MPPs into lethally irradiated host mice.  Bone marrow cells were harvested from reconstituted mice 6 days after injection and analyzed by FACS (figure 4A).  The results show that phenotypic CMPs were only detected in hosts given Flt3(low)VCAM-1+ MPPs. 

      Based on the results obtained in various lineage differentiation tests, Lai et al. determined an alternate pathway for HSC lineage commitment.  The new hierarchy of HSC differentiation – constructed by the authors of this study – challenges the classic model of HSC differentiation, which states the first step of HSC lineage restriction is of lymphoid versus myeloid fate.  Lai et al. believe that lineage restriction in hematopoiesis occurs first in Flt3(low)VCAM-1+ MPPs which lose their self-renewal ability, but are still able to differentiate into lymphoid or myeloid lineages. 

Comments 

      The observations and results presented in the article by Lai et al. challenge the traditional model of HSC hierarchy and lineage restriction.  This study suggests that Flt3(low)VCAM-1+ MPPs are the most primitive type of MPP and thus give rise to all other HSC progenitors: Flt3(high)VCAM-1+ MPP, Flt3(high)VCAM-1- MPP, and CMPs.  The study of HSC differentiation conducted by Lai et al. therefore follows the monophyletic hypothesis, which suggests both lymphoid and myeloid lineages arise via a common stem cell.  Further studies must be conducted to determine how the asymmetric model of lineage divergence is coordinated.