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Adult murine hematopoietic stem cells and progenitors: an update on their identities, functions, and assays

  • Louise E. Purton
    Correspondence
    Offprint requests to: Louise E. Purton, St Vincent's Institute of Medical Research, 9 Princes St, Fitzroy 3065, Victoria, Australia.
    Affiliations
    St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia; Department of Medicine, The University of Melbourne, Parkville, Victoria, Australia
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Open AccessPublished:October 22, 2022DOI:https://doi.org/10.1016/j.exphem.2022.10.005

      Highlights

      • This is a review of murine HSCs and new HSC assays developed in the past 15 years.
      • This review summarizes the immunophenotypes and functions of HSCs and MPPs.
      • This review provides a summary of studies of platelet/myeloid-biased and lymphoid-biased HSCs.
      The founder of all blood cells are hematopoietic stem cells (HSCs), which are rare stem cells that undergo key cell fate decisions to self-renew to generate more HSCs or to differentiate progressively into a hierarchy of different immature hematopoietic cell types to ultimately produce mature blood cells. These decisions are influenced both intrinsically and extrinsically, the latter by microenvironment cells in the bone marrow (BM). In recent decades, notable progress in our ability to identify, isolate, and study key properties of adult murine HSCs and multipotent progenitor (MPP) cells has challenged our prior understanding of the hierarchy of these primitive hematopoietic cells. These studies have revealed the existence of at least two distinct HSC types in adults: one that generates all hematopoietic cell lineages with almost equal potency and one that is platelet/myeloid-biased and increases with aging. These studies have also revealed distinct MPP cell types that have different functional potential. This review provides an update to these murine HSCs and MPP cells, their key functional properties, and the assays that have been used to assess their potential.
      Our knowledge of hematopoiesis has significantly advanced in recent decades largely because of improved technology. The development of and subsequent improvements in the capacity of flow cytometry–based applications have enabled researchers to identify, isolate, and study populations of hematopoietic cells, including hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). In 2007, we published an extensive review of methods used to isolate and assay murine HSCs and MPPs [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ]. Since then, there have been significant advances in flow cytometry–based methods to purify and study the function of distinct populations of HSCs and MPPs. This review provides an update to our previous one and provides an overview of some of these recent studies. For those who are new to the field, our previous review [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ] discusses many different assays used to study HSCs and MPPs, and it is recommended as a companion reading to this review.
      In this review, long-term repopulating HSCs (LT-HSCs) refer to multilineage repopulating cells that can generate blood cells for at least 16 weeks after transplantation in recipient mice. Short-term repopulating HSCs (ST-HSCs) are cells that can repopulate mice for at least 12 weeks after transplantation. MPPs are cells that have diminished in vivo repopulating activity compared with both LT-HSCs and ST-HSCs and have varying multipotent potential, depending on the MPP subtype. All studies referenced in this review have performed relevant in vivo repopulating assays to assess the repopulating potential of HSC and MPP populations.
      Some of the studies discussed in this review have assessed the in vivo repopulation potential of single cells, which, although technically challenging and requires larger cohorts of recipient mice, is the most stringent test for evaluating HSC/MPP repopulating capacity and enables assessment of the purity of the population identified by fluorescent cell surface markers or reporter mice. However, note that despite these recent advances, the majority, if not all, of the HSC and MPP populations described here rely heavily on immunophenotype. Furthermore, although the function of HSCs has been assessed using single-cell transplants in some studies, the heterogeneous behavior of the HSCs being assessed in these studies suggests that we have not yet achieved 100% pure populations of HSCs (and likely MPPs) using any of the current standard approaches.

      HSC POPULATIONS IDENTIFIED USING DIFFERENT COMBINATIONS OF SLAM, CD34, AND CD135 MARKERS

      In our previous review, cell surface markers CD34 and CD135 (FLT3) had been used by the Nakauchi and Jacobsen laboratories to identify LT-HSCs (LKS+ CD34− CD135−), ST-HSCs (LKS+ CD34+ CD135−), and a population that consisted of transiently repopulating cells termed MPP cells (LKS+ CD34+ CD135+) [
      • Adolfsson J
      • Månsson R
      • Buza-Vidas N
      • et al.
      Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment.
      ,
      • Yang L
      • Bryder D
      • Adolfsson J
      • et al.
      Identification of Lin(-)Sca1(+)kit(+)CD34(+)Flt3- short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients.
      ,
      • Osawa M
      • Hanada K
      • Hamada H
      • Nakauchi H.
      Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.
      ]. The Morrison group had also reported a method that isolated HSCs on the basis of the expression of CD150 and CD48 (the SLAM markers), with CD150+ CD48− CD41− cells representing the most primitive cell population [
      • Kiel MJ
      • Yilmaz OH
      • Iwashita T
      • Yilmaz OH
      • Terhorst C
      • Morrison SJ.
      SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.
      ,
      • Yilmaz OH
      • Kiel MJ
      • Morrison SJ.
      SLAM family markers are conserved among hematopoietic stem cells from old and reconstituted mice and markedly increase their purity.
      ]. Combinations of the SLAM markers (CD150 and CD48), CD34 and/or CD135 have now been used to further purify HSC and MPP populations within lineage-negative, c-KIT+, Sca-1+ (LKS+) cells (also commonly referred to as LSK).
      Two separate studies from the Trumpp laboratory identified and explored the functional potential of populations that differentially expressed CD150, CD48, CD135 and CD34 markers within LKS+ cells. In the first study, Wilson et al. [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ] showed that differential expressions of the CD150, CD48, CD135, and CD34 markers within LKS+ identified five populations that were termed HSC (LKS+ CD150+ CD48− CD34− CD135−), MPP1 (LKS+ CD150+ CD48–CD34+ CD135–), MPP2 (LKS+ CD150+ CD48+ CD34+ CD135–), MPP3 (LKS+ CD150− CD48+ CD34+ CD135−), and MPP4 (LKS+ CD150− CD48+ CD34+ CD135+). A series of elegant studies showed that the HSC population was highly enriched for dormant (very quiescent) HSCs. The HSCs were shown to be activated after hematopoietic stress conditions (treatment of mice with either the chemotherapy agent, 5-fluorouracil, or granulocyte colony-stimulating factor) to self-renew and replenish hematopoiesis and could then return to dormancy [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ].
      In the second study from the Trumpp group, Cabezas-Wallscheid et al. [
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ] further explored the functional potential of the HSC and MPP populations identified by Wilson et al. [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ]. They also provided a comprehensive proteome, transcriptome, and DNA methylome resource of the HSCs and MPPs [
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ]. Among the findings of this resource was the enrichment of retinoic acid signaling pathway genes in HSCs. In a subsequent study, they showed that the biologically active vitamin A derivative, all-trans retinoic acid (ATRA), was important in regulating HSC dormancy and self-renewal [
      • Cabezas-Wallscheid N
      • Buettner F
      • Sommerkamp P
      • et al.
      Vitamin A-retinoic acid signaling regulates hematopoietic stem cell dormancy.
      ]. These studies independently confirmed the findings of our previous studies that showed that ATRA is a key regulator of murine HSCs and that ATRA treatment increases the serial transplantability of cultured murine HSCs [
      • Purton LE
      • Bernstein ID
      • Collins SJ.
      All-trans retinoic acid enhances the long-term repopulating activity of cultured hematopoietic stem cells.
      ,
      • Purton LE
      • Bernstein ID
      • Collins SJ.
      All-trans retinoic acid delays the differentiation of primitive hematopoietic precursors (lin-c-kit+Sca-1(+)) while enhancing the terminal maturation of committed granulocyte/monocyte progenitors.
      ,
      • Purton LE
      • Dworkin S
      • Olsen GH
      • et al.
      RAR{gamma} is critical for maintaining a balance between hematopoietic stem cell self-renewal and differentiation.
      ].
      The studies from the Passegue laboratory also used a combination of SLAM and CD135 markers in LKS+ cells to define LT-HSC (LKS+ CD150+ CD48− CD135−), ST-HSC (LKS+ CD150− CD48− CD135−), and three MPP types: MPP2 (LKS+ CD150+ CD48+ CD135−), MPP3 (LKS+ CD150− CD48+ CD135−), and MPP4 (LKS+ CD150− CD48+ CD135+) [
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ]. These studies did not incorporate CD34; hence, the LT-HSC identified by Pietras et al. [
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ] comprised both the HSC and MPP1 cells separately identified and investigated by the Trumpp laboratory [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ,
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ]. The ST-HSCs were a new population that had not been investigated in the other studies. In contrast, the studies by the Trumpp group revealed that all of the MPP2–MPP4 cells expressed CD34, which was confirmed in the study by Pietras et al. [
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ]; hence, the properties of these cell types can be directly compared.
      Collectively, the studies from the Trumpp and Passegue laboratories concluded that HSCs and ST-HSCs are multipotent HSCs, MPP2 cells are megakaryocyte-biased progenitors, MPP3 cells are myeloid-biased progenitors, and MPP4 cells are lymphoid-biased progenitors [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ,
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ,
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ]. The functional properties of each of these cell types were also investigated using in vitro and in vivo studies and are summarized in Table 1.
      Table 1Properties of HSC and progenitor cell types isolated based on SLAM, CD34, and CD135 markers
      Original nameLT-HSC (Trumpp laboratory) [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ,
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ]
      LT-HSC (Passegue laboratory)
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      MPP1 [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ,
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ]
      ST-HSC
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      MPP2 [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ,
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ,
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ]
      MPP3 [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ,
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ,
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ]
      MPP4 or LMPP [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ,
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ,
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ]
      Revised name
      • Challen GA
      • Pietras EM
      • Wallscheid NC
      • Signer RAJ.
      Simplified murine multipotent progenitor isolation scheme: establishing a consensus approach for multipotent progenitor identification.
      Dormant HSCLT-HSCActive HSCMPPMPPMK/EMPPG/MMPPLy
      Flow cytometry gating strategyLKS+ CD150+ CD48− CD34− CD135−LKS+ CD150+ CD48− CD34+/− CD135−LKS+ CD150+ CD48− CD34+ CD135−LKS+ CD150− CD48− CD135−LKS+ CD150+ CD48+ CD34+ CD135−LKS+ CD150− CD48+ CD34+ CD135−LKS+ CD150− CD48+ CD34+ CD135+
      Primary transplant

      Competitive repopulation potential when cotransplanted with 2 × 105 wild-type BM cells (0.2% donor cells considered positive)
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      .
      50 HSC: multilineage, 100% recipients repopulated from 50 HSC at 16 weeks after transplant [
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ].

      2,000 HSC: donor contribution of approximately 56% (T lymphocytes), 70% (B lymphocytes), 82% (myeloid cells) at 16 weeks after transplant
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      .
      ND50 MPP1: 56% of recipients multilineage at 16 weeks after transplant
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      .
      ND2,000 MPP2: Rapid myeloid reconstitution, donor contribution of approximately 23% (T lymphocytes), 35% (B lymphocytes), 32% (myeloid cells) at 16 weeks after transplant
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      .
      2,000 MPP3: Myeloid repopulation between 1 and 3 weeks after transplant, less than 5% multilineage repopulating donor cells at 9–16 weeks after transplant
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      .
      2,000 MPP4: Transient repopulation of B lymphocytes, less than 1% multilineage donor cell reconstitution at 16 weeks after transplant
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      .
      Secondary transplant potential (1 ×106 BM cells transplanted from primary recipients of 50 cells and 2 × 105 wild-type BM cells,≥0.2% donor cells considered positive)
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      .
      8/10 recipients Donor cells detected at 8 weeks after transplant.ND0 recipients had detectable donor cells at 8 weeks after transplant.NDNDNDND
      Competitive repopulation potential when 50 cells were cotransplanted with 3 × 105 Sca-1-depleted BM cells (only the average % reconstitution was provided for each group)
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      .
      NDAt 4 weeks after transplant: similar donor reconstitution to ST-HSCs, superior myeloid engraftment.

      At 16 weeks after transplant: approximately 40% multilineage donor cell reconstitution.
      NDAt 4 weeks after transplant: similar donor cell reconstitution to LT-HSCs, superior lymphoid engraftment.

      At 16 weeks after transplant: approximately 5% donor cells, exclusively lymphoid.
      NDNDND
      Cell cycle
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      70% in G0, <2% actively cycling (S+G2/M)ND39% in G0, 14% actively cycling (S+G2/M)ND<20% in G0,, ∼25% actively cycling (S+G2/M)<10% in G0, ∼24% actively cycling (S+G2/M)<5% in G0, ∼28% actively cycling (S+G2/M)
      Label retaining cells measured at 70 days of chase after 10–13 days of treatment with BrdU32.7 ± 4.7% (and approximately 5% after 306 days of chase)ND∼10%ND∼7%∼5%<2%
      BrdU incorporation after 1 hour pulse in vitro
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ND<10%[
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ]
      ND<10%>20%>20%>20% (slightly less than MPP2 and MPP3)
      Proliferative potential during 10 days of liquid suspension culture
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      NDHighNDHighLower than LT-HSC and ST-HSC, higher than MPP3 and MPP4Lower than LT-HSC, ST-HSC and MPP2, higher than MPP4Lowest
      CFC potential from single-sorted cells
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      NDHighest plating efficiency, high proportion of Meg/E CFCsNDHighest plating efficiencyLower plating efficiency than LT-HSC and ST-HSC, high proportion of Meg/E CFCsLower plating efficiency than LT-HSC and ST-HSCLowest plating efficiency
      Day 12 CFU-S
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ND1/20ND1/201/801/801/250
      B lymphocyte production in OP9 cocultures
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      NDDetectable by day 21 of cocultureNDND1/311/401/5
      Immature T lymphocyte production (DN2= CD44+CD25+, DN3= CD44-CD25+) in OP9-DL1 cocultures during 12 days
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      NDYes, produced some DN2 and DN3 at day 12, very minimal at day 8NDNDYes, produced DN2 but few DN3, minimal at day 8Yes, produced more than all other types except MPP4, produces DN2 at day 8, DN2 and DN3 at 12 daysYes, most robust, produced DN2 and DN3 within 8 days, more DN3 at 12 days
      CD41 cell surface expression
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      NDMajority of cells + (bright)NDTwo populations: one with low expression (less than 50% of cells), the other negative expressionMajority of cells + (bright)Two populations: one with low expression (more than 50% of cells), the other negative expressionHomogenously low/negative
      Vwf-GFP expression
      • Morita Y
      • Ema H
      • Nakauchi H
      Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.
      60%NDNDNDNDND0.22%
      ESAM cell surface expression
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      NDHighNDHighTwo populations: one high (majority of cells), the other low/negativeTwo populations: one high (majority of cells), the other low/negativeTwo populations: one low/negative (majority of cells), the other intermediate
      BM = bone marrow; BrdU = bromodeoxyuridine; CFC = colony-forming cell; CFU-S = colony-forming unit-spleen; DN = double negative; G/M = granulocyte/monocyte-biased; HSC = hematopoietic stem cell; LT-HSC = long-term repopulating hematopoietic stem cell; Ly = lymphocyte-biased; MPP = multipotent progenitor; LMPP = lymphoid-primed multipotent progenitor; MK/E = megakaryocyte/erythroid-biased; ND = not determined; ST-HSC = short-term repopulating hematopoietic stem cell.
      Sommerkamp et al. [
      • Sommerkamp P
      • Romero-Mulero MC
      • Narr A
      • et al.
      Mouse multipotent progenitor 5 cells are located at the interphase between hematopoietic stem and progenitor cells.
      ] recently identified two distinct populations within the ST-HSCs on the basis of their expression of CD34, with CD34+ ST-HSCs being termed MPP5 and CD34− ST-HSCs being termed MPP6 [
      • Sommerkamp P
      • Romero-Mulero MC
      • Narr A
      • et al.
      Mouse multipotent progenitor 5 cells are located at the interphase between hematopoietic stem and progenitor cells.
      ]. When 2,000 cells of either type were transplanted together with 2 × 105 competing spleen cells into lethally irradiated recipients, both populations showed multilineage reconstitution, although MPP6 showed increased reconstitution of myeloid cells compared with MPP5 [
      • Sommerkamp P
      • Romero-Mulero MC
      • Narr A
      • et al.
      Mouse multipotent progenitor 5 cells are located at the interphase between hematopoietic stem and progenitor cells.
      ]. Repopulation was not assessed beyond 12 weeks, and no other functional data were provided for MPP6; hence, these populations are not included in Table 1 or discussed further in this review.
      In order to consolidate the findings of these independent laboratories and avoid terminology confusion, MPP populations were recently summarized and reclassified by Challen et al. [
      • Challen GA
      • Pietras EM
      • Wallscheid NC
      • Signer RAJ.
      Simplified murine multipotent progenitor isolation scheme: establishing a consensus approach for multipotent progenitor identification.
      ]. MPP2 has been reclassified as MPPMK/E (megakaryocyte/erythroid-biased), MPP3 has been reclassified as MPPG/M (granulocyte/monocyte-biased), and MPP4 has been reclassified as MPPLy (lymphocyte-biased). They also suggested renaming ST-HSCs to MPPs (referring to MPPs with unbiased multilineage reconstituting potential) because the authors defined HSCs as cells that have long-term repopulating (and therefore, self-renewal) potential. However, the classical definition of ST-HSCs are cells that show multilineage repopulation in mice for up to 12 weeks after transplantation [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ,
      • Purton LE
      • Dworkin S
      • Olsen GH
      • et al.
      RAR{gamma} is critical for maintaining a balance between hematopoietic stem cell self-renewal and differentiation.
      ]. Interestingly, both the ST-HSC and MPP2 populations currently meet those criteria and are worthy of further investigation to conclusively identify whether they are ST-HSCs or MPPs.

      IDENTIFICATION OF PLATELET/MYELOID-BIASED AND LYMPHOID-BIASED HSCs: A NEW CLASSIFICATION OF THE HSC HIERARCHY

      It has long been recognized that HSCs do not all behave the same on transplantation into irradiated mice; however, it was unclear as to why this was the case. A seminal transplantation study of single HSCs by the Eaves group [
      • Dykstra B
      • Kent D
      • Bowie M
      • et al.
      Long-term propagation of distinct hematopoietic differentiation programs in vivo.
      ] used a population of lineage-negative bone marrow (BM) cells further purified on the basis of their intermediate cell surface expression of CD45 and their low intensity of fluorescent dyes rhodamine123 and Hoescht 33342 (the use of each of these dyes to isolate HSCs was reviewed previously by Purton et al [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ]). Transplantation of single cells into sublethally irradiated W41/W41 mice (which are c-KIT deficient and engraft more readily with very low numbers of HSCs [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ,
      • Trevisan M
      • Yan XQ
      • Iscove NN.
      Cycle initiation and colony formation in culture by murine marrow cells with long-term reconstituting potential in vivo.
      ]) identified that there were four distinct types of murine repopulating cells classified on the basis of their potential to produce myeloid (granulocytes/monocytes), B lymphocytes, and/or T lymphocytes in the mice for up to 24 weeks after transplant. The α-cells were myeloid-biased; β-cells had a balanced production of myeloid cells, B lymphocytes, and T lymphocytes; γ-cells were lymphoid-biased, producing both B and T lymphocytes to similar proportions, with limited myeloid potential; and the δ-cells had a restricted production of T lymphocytes. Note that contribution to platelets could not be assessed in these studies because platelets do not express CD45.1 or CD45.2, which are routinely used to distinguish donor, host, and competing cells in studies that use mice generated on the C57BL/6 background [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ].

      E-SLAM HSCs

      The Eaves group further showed that the endothelial protein C receptor (EPCR) could be used to identify HSCs in both E14.5 fetal liver (FL) and adult BM [
      • Kent DG
      • Copley MR
      • Benz C
      • et al.
      Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential.
      ]. Transplantation studies of single cells into sublethally irradiated W41/W41 mice revealed that cells that were defined as CD45+, EPCR+, CD48– and CD150+ (E-SLAM HSCs) were the most primitive and were enriched for cells with high self-renewal (SR) capacity (high SR E-SLAM HSCs). From a total of 62 mice that received a single-cell transplantation of CD45+ EPCR+ CD48− CD150+ E-SLAM HSCs, 43% of the single cells had high SR, 13% had low SR, and 2% were short-term repopulating stem cells, with 42% lacking repopulation potential at or beyond 8 weeks after transplantation. By contrast, of the 28 recipients of CD45+ EPCR+CD48− CD150− cells, 7% had high SR, 32% had low SR, 18% were short-term repopulating cells, and 43% lacked repopulation potential. These CD45+ EPCR+CD48− CD150− cells were therefore enriched for low SR potential (low SR E-SLAM HSCs).
      Kent et al. [
      • Kent DG
      • Copley MR
      • Benz C
      • et al.
      Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential.
      ] performed quantitative polymerase chain reaction (qPCR) studies to compare the expression of transcripts in CD45+ EPCR+ CD48− CD150+ E-SLAM HSCs and CD45+ EPCR+ CD48− CD150− cells. The E-SLAM HSCs were shown to express notably higher levels of transcripts of genes that have previously been shown to regulate HSC SR: Prnp [
      • Zhang CC
      • Steele AD
      • Lindquist S
      • Lodish HF.
      Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal.
      ], Gata3 [
      • Pandolfi PP
      • Roth ME
      • Karis A
      • et al.
      Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis.
      ], and Bmi1 [
      • Park IK
      • Qian D
      • Kiel M
      • et al.
      Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells.
      ,
      • Lessard J
      • Sauvageau G.
      Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells.
      ]. In a search for new regulators of HSC SR, they constructed LongSAGE libraries generated from E14.5 FL and adult BM HSCs, with gene candidates further narrowed by a comparison with previously published microarrays of these HSC populations. This approach identified 27 candidate genes, nine of which were consistently upregulated in E14.5 FL and BM E-SLAM cells. qPCR studies revealed that transcripts of four of these genes were significantly increased in high SR E-SLAM BM HSCs compared with low SR E-SLAM BM HSCs: Smarcc2, Rhob, Pld3, and Vwf [
      • Kent DG
      • Copley MR
      • Benz C
      • et al.
      Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential.
      ]. Key functional properties of the high SR and low SR E-SLAM HSCs from adult BM are summarized in Table 2.
      Table 2Properties of HSCs identified using EPCR
      Original nameE-SLAM HSCs
      • Kent DG
      • Copley MR
      • Benz C
      • et al.
      Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential.
      CD45+ EPCR+ CD48− CD150− cells
      • Kent DG
      • Copley MR
      • Benz C
      • et al.
      Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential.
      Revised nameHigh SR E-SLAMLow SR E-SLAM
      Flow cytometry gating strategyCD45+ EPCR+ CD48− CD150+CD45+ EPCR+ CD48− CD150−
      Single-cell transplants assessed at 4 months after transplant into W41/W41 mice

      43% high SR

      13% low SR

      2% STRC

      42% non-RC

      56% of population estimated to be an HSC
      7% high SR

      32% low SR

      18% STRC

      43% non-RC

      39% of population estimated to be an HSC
      Lineage compositions of repopulated mice from single a HSC

      α = myeloid-biased,

      β = balanced myeloid and lymphoid,

      γ = lymphoid-biased (B and T lymphocytes),

      δ = restricted production of T lymphocytes

      ∼16% α

      ∼61% β

      ∼22% γ

      0% δ

      E-SLAM HSCs repopulated secondary recipients (reported in the text)
      0% α

      ∼18% β

      ∼45% γ

      ∼36% δ

      Frequency of LTC-IC from single cells43%7%
      EPCR = endothelial protein C receptor; HSC = hematopoietic stem cell; LTC-IC= long-term culture-initiating cell; RC = repopulating cell; SR = self-renewal; STRC= short-term repopulating cell.

      CD150HIGH MYELOID-BIASED HSCs AND PROGENITORS

      The Nakauchi laboratory screened 118 different cell surface markers by flow cytometry to further purify HSCs within LKS+ CD34− HSCs [
      • Osawa M
      • Hanada K
      • Hamada H
      • Nakauchi H.
      Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.
      ]. They showed that the LKS+ CD34− HSCs could be subdivided into three fractions on the basis of their expression of CD150 (high, medium, or negative) and termed them CD150high (which were also shown to express high levels of CD38), CD150med, and CD150neg HSCs [
      • Morita Y
      • Ema H
      • Nakauchi H
      Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.
      ]. Initial competitive repopulation studies of 10 HSCs competed against 2 × 105 wild-type BM cells revealed that the CD150high HSCs had low peripheral blood chimerism (<25% donor cells) at 2 months after transplantation, however, this increased by 5 months after transplantation. These HSCs were myeloid-biased but did repopulate the lymphoid lineages. The CD150med HSCs showed relatively stable reconstitution at all time points and were lymphoid-biased, but most recipients showed myeloid cell repopulation, and the average donor cell reconstitution was higher than that from both the CD150high and CD150neg HSCs. By contrast, the CD150neg HSCs had the poorest reconstitution potential and were lymphoid-biased with inferior myeloid repopulating potential compared with that of the CD150med HSCs [
      • Morita Y
      • Ema H
      • Nakauchi H
      Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.
      ].
      Competitive transplantation studies of single HSCs confirmed the observations of the competitive transplants of 10 HSCs and revealed that the CD150high HSCs had the highest repopulating capacity in secondary recipients [
      • Morita Y
      • Ema H
      • Nakauchi H
      Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.
      ]. Furthermore, single CD150neg HSCs had the poorest secondary transplantation potential. Key functional properties of these studies by Morita et al. [
      • Morita Y
      • Ema H
      • Nakauchi H
      Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.
      ] are summarized in Table 3.
      Table 3Properties of HSCs identified using CD150 expression within the LKS+ CD34− population
      Original nameCD150 high
      • Morita Y
      • Ema H
      • Nakauchi H
      Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.
      CD150 medium
      • Morita Y
      • Ema H
      • Nakauchi H
      Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.
      CD150 negative
      • Morita Y
      • Ema H
      • Nakauchi H
      Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.
      Revised nameCD150highCD150medCD150neg
      Flow cytometry gating strategyLKS+ CD34− CD150highLKS+ CD34− CD150medLKS+ CD34− CD150neg
      3 primary recipients died before secondary transplantation.
      tive
      Primary transplant

      Competitive repopulation potential

      10 HSCs cotransplanted with 2 × 105 wild-type BM cells (≥0.3% donor cells at one or more time point after transplant, monitored up to 5 months after transplant)
      19 recipients showed long-term repopulation, myeloid-biased but repopulated all lineages

      Chimerism was low in 15/19 recipients at early time points and increased during the 5 months after transplant, but remained lower, on average, than CD150med HSCs

      19 recipients showed long-term repopulation, lymphoid-biased but repopulated all lineages

      Chimerism was relatively stable and high (median chimerism ∼25%) in 11/19 recipients during the 5 months after transplant

      14 recipients showed long-term repopulation, lymphoid-biased, poorer reconstitution of myeloid cells compared with CD150med HSCs

      Chimerism was relatively stable and low in 11/19 recipients during the 5 months after transplant with the exception of one recipient, which showed increased chimerism after transplant, achieving approximately 75% chimerism

      Primary transplant

      Competitive repopulation potential

      Single HSCs cotransplanted with 2 × 105 wild-type BM cells (≥0.3% donor cells at one or more time point after transplant, monitored up to 5 months after transplant)

      16/40 recipients showed long-term repopulation, myeloid-biased but repopulated all lineages with the exception of 2/20 mice which showed low levels of myeloid cell reconstitution

      13/40 recipients showed long-term repopulation, lymphoid-biased but repopulated all lineages

      13/40 recipients showed long-term repopulation, lymphoid-biased, poorer reconstitution of myeloid cells compared with CD150med HSCs

      1/40 recipients repopulated only T lymphocytes

      Secondary transplant

      5 × 106 BM cells transplanted from primary recipients of single HSCs competed against 2 × 105 wild-type BM cells (≥0.3% donor cells at one or more time point after transplant, monitored up to 5 months after transplant)

      13/13
      3 primary recipients died before secondary transplantation.
      recipients showed long-term repopulation, these were myeloid-biased but most recipients had lymphoid reconstitution

      1/10 HSCs were identified to be latent HSCs that showed poor myeloid reconstitution in primary recipients and robust reconstitution in secondary recipients

      1/20 cells identified as myeloid-restricted progenitors

      4/13
      1 primary recipient died before secondary transplantation.
      recipients showed long-term repopulation. 2 recipients were lymphoid-restricted, two had myeloid-biased multilineage repopulation
      2/14
      2 primary recipients died before secondary transplantation.
      recipients showed long-term repopulation, all were lymphoid-biased with minimal myeloid reconstitution

      5/12 recipients repopulated only T lymphocytes

      Single-cell colony assays

      Approximately 42/48 single HSCs formed colonies

      Colony composition

      ∼60% nmEM

      ∼ 7% nmM

      ∼ 12% nm

      <1% m

      Remainder defined as blast-like or “other” colonies
      Approximately 45/48 single HSCs formed colonies

      Colony composition

      ∼30% nmEM

      ∼ 20% nmM

      ∼ 40% nm

      ∼4% m

      Remainder defined as blast-like or “other” colonies
      Approximately 41/48 single HSCs formed colonies

      Colony composition

      ∼7% nmEM

      ∼ 14% nmM

      ∼ 66% nm

      ∼7% m

      Remainder defined as blast-like or “other” colonies
      BM = bone marrow; HSC = hematopoietic stem cell; m = macrophage colonies; nm = neutrophil/macrophage colonies; nmEM = neutrophil/macrophage/erythroblast/megakaryocyte colonies; nmM = neutrophil/macrophage/megakaryocyte colonies.
      a 3 primary recipients died before secondary transplantation.
      b 1 primary recipient died before secondary transplantation.
      c 2 primary recipients died before secondary transplantation.
      These single HSC studies also suggested the existence of a latent HSC (estimated to be approximately 1/10 CD150high HSCs) [
      • Morita Y
      • Ema H
      • Nakauchi H
      Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.
      ]. The latent HSC produced virtually undetectable levels of blood cells for approximately 12 weeks after transplantation at or after which they contributed to low levels of myeloid cells. Intriguingly, when BM cells from these primary recipients were transplanted into secondary recipient mice, they robustly reconstituted all cell lineages assessed (myeloid, T lymphoid, and B lymphoid). Furthermore, approximately 1/20 CD150high HSCs showed a low level of myeloid reconstitution and no lymphoid reconstitution in both primary and secondary recipients [
      • Morita Y
      • Ema H
      • Nakauchi H
      Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.
      ]. A subsequent study revealed that these cells were myeloid-restricted progenitors (MyRPs) that lack SR potential and are formed by asymmetric divisions of HSCs [
      • Yamamoto R
      • Morita Y
      • Ooehara J
      • et al.
      Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.
      ].
      A limitation of these prior studies was the inability to detect chimerism in the erythroid and platelet lineages. To enable this, they generated Kusabira Orange (KuO) fluorescent reporter mice [
      • Yamamoto R
      • Wilkinson AC
      • Ooehara J
      • et al.
      Large-scale clonal analysis resolves aging of the mouse hematopoietic stem cell compartment.
      ]. Single-cell transplantation studies confirmed that the latent HSCs could repopulate all cell lineages. The effects of aging were also assessed in these studies but are not discussed further in this review, which focuses largely on properties of young HSCs.

      Vwf-EXPRESSING HSCs

      The Jacobsen and Nerlov laboratories generated CD45.2+ Vwf-eGFP BAC mice and used them to demonstrate that approximately 60% of adult HSCs defined as LKS+ CD150+ CD48− CD34− cells (which are similar to the HSC population described and characterized by the Trumpp laboratory [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ,
      • Cabezas-Wallscheid N
      • Klimmeck D
      • Hansson J
      • et al.
      Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis.
      ]) were Vwf-GFP+ [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ]. By contrast, the LKS+ CD150− CD135+ cells (akin to MPP4/MPPLy) did not express Vwf-GFP. Furthermore, 96% of FL HSCs (identified as being LKS+ CD150+ CD48–) were Vwf-GFP+ [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ]. These studies confirmed the observations of Kent et al. [
      • Kent DG
      • Copley MR
      • Benz C
      • et al.
      Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential.
      ] that Vwf may have a role in HSC SR.
      When 10 Vwf-GFP+ HSCs were transplanted together with 2 × 105 wild-type (WT) CD45.1+ competing BM cells, 15/48 recipients had notable donor cell reconstitution (defined as >1% contribution to at least one of the lineages at both 10 and 16 weeks after transplantation). Of these 15 mice, there was a variable contribution to the different cell lineages at 16 weeks after transplantation, although all recipients had platelet reconstitution. By contrast, 10 of 42 of the Vwf-GFP− HSCs showed notable donor cell reconstitution, and these were lymphoid-biased, although they did contribute to the other cell lineages, including platelets.
      Single HSCs were transplanted together with 1 × 106 W41/W41 CD45.1+ competing BM cells. In these initial studies, only 3 of 17 recipients showed detectable reconstitution, and all recipients had platelet-biased reconstitution, with minimal detection of B and T lymphocytes [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ]. Additional transplant studies suggested that the Vwf-GFP+ HSCs (termed platelet/biased HSCs) were at the top of the HSC hierarchy and generated Vwf-GFP− HSCs, whereas Vwf-GFP− HSCs did not robustly generate Vwf-GFP+ HSCs [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ]. Note that it is currently unclear whether these studies have revealed that Vwf-GFP+ HSCs show a myeloid/platelet-bias versus myeloid/platelet-restriction. This is an important question in the HSC field, which requires further clarification in future studies.
      Microarray studies revealed that Vwf-GFP+ HSCs had higher expression of megakaryocyte lineage genes compared with Vwf-GFP− HSCs, including Clu, Gpr64, Sdpr, Mpl, and Zfpm1, in addition to genes associated with bipotent megakaryocyte/erythroid progenitors [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ]. Multiplex single-cell qPCR of Vwf-GFP+ and Vwf-GFP− LKS+ CD34− CD150+ CD48− HSCs confirmed expression of these transcripts in most single cells [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ].
      More recently, the Jacobsen and Nerlov laboratories generated Vwf-tdTomato mice, providing a brighter fluorescent reporter mouse to reliably detect the repopulation of single Vwf-HSCs [
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ]. Single HSCs (LKS+ CD34–CD150+ CD48− Vwf-tdTomato+) were transplanted together with 2 × 105 CD45.1+ WT BM cells (58 recipients) or 1 × 106 W41/W41 CD45.1+ competing BM cells (292 recipients). Comparable results were obtained for both competitive transplant types in terms of the donor cell reconstitution. Approximately 40% of recipients showed 0.1% donor cell contribution to at least one lineage at 16–18 weeks after transplantation, with almost 50% of these recipients showing repopulation in all lineages (platelets, erythrocytes, myeloid, B lymphocytes, and T lymphocytes). Consistent with their previous publication, they observed different proportions of donor cell contribution to the lineages; however, all HSCs contributed to platelet reconstitution [
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ].
      Interestingly, although there was evidence of lineage bias in vivo, when single Vwf-tdTomato+ LT-HSCs were isolated from recipient mice that exclusively produced platelets, the HSCs were able to generate other hematopoietic lineages (granulocytes, monocyte/macrophages, and T lymphocytes) in vitro, and gene expression studies of single HSCs indicated that they expressed transcripts associated with those lineages [
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ]. Collectively, these studies suggested that, irrespective of their in vivo repopulating potential, all Vwf-tdTomato+ LT-HSCs retained multipotency. A summary of properties of the platelet-biased and lymphoid-biased HSCs isolated using Vwf-reporter mice is in provided in Table 4.
      Table 4Properties of HSCs identified using vWF-reporter mice
      Original nameVwf-GFP+ platelet-biased HSC [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ,
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ]
      Vwf-GFP- lymphoid-biased HSC [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ,
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ]
      Revised nameVwf+ HSCVwf- HSC
      Flow cytometry gating strategyLKS+ CD150+ CD48− CD34−Vwf+ identified using either Vwf-GFP or Vwf-tdTomato reporter mice

      LKS+ CD150+ CD48− CD34−Vwf- identified using either Vwf-GFP or Vwf-tdTomato reporter mice
      Primary transplant

      Competitive repopulation potential

      10 HSCs cotransplanted with 2 × 105 wild-type BM cells (1% donor cells positive at both 10 and 16 weeks after transplant)

      Biased reconstitution defined when cells were repopulated >50% higher than the other lineages
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      15/48 recipients had repopulation in at least one lineage, all recipients repopulated platelets

      5/15 were platelet-biased

      4/15 were platelet/myeloid-biased

      1/15 was myeloid-biased

      2/15 were balanced

      3/15 were lymphoid-biased
      10/42 recipients repopulated mice, all were lymphoid-biased but did contribute to the other lineages
      Primary transplant

      Competitive repopulation potential

      1 HSC cotransplanted with 2 × 105 wild-type BM cells (0.1% donor cells considered positive at 16–18 weeks after transplant)
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      25/58 recipients had detectable donor cells in at least one lineage

      3/58 recipients were platelet-biased

      5/58 recipients were platelet/erythroid/myeloid-biased

      6/58 recipients were platelet/erythroid/myeloid/B lymphocyte restricted

      11/58 recipients repopulated all lineages
      ND
      Primary transplant

      Competitive repopulation potential when single HSCs cotransplanted with 1 × 106 W41/W41 BM cells (0.1% donor cells considered positive at 16–18 weeks after transplant)
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      109/292 recipients had detectable donor cells in at least one lineage

      12/292 recipients were platelet-biased

      3/292 recipients were platelet/erythroid-biased

      18/292 recipients were platelet/erythroid/myeloid-biased

      54/292 recipients repopulated all lineages
      ND
      Potential to form other HSC typeProduced high numbers of Vwf-GFP- HSCs at 7 and 32 weeks after transplantMinimal numbers of Vwf-GFP+ HSCs produced at 7 and 32 weeks after transplant
      CFC potential from single-sorted cellsHigh cloning efficiency, including large proportions containing megakaryocytesHigh cloning efficiency, fewer megakaryocytes
      BM = bone marrow; CFC = colony-forming cell; HSC = hematopoietic stem cell.
      Recognizing that not all laboratories can access Vwf-reporter mice, Carrehla et al. [
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ] searched for flow cytometry markers that could be used instead. The Morrison laboratory had previously reported that LKS+ CD150+ CD48− HSCs lack expression of CD229 [
      • Oguro H
      • Ding L
      • Morrison SJ.
      SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.
      ]. Carrehla et al. [
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ] performed single-HSC transplants to identify that platelet-biased Vwf-tdTomato+ HSCs were LKS+ CD150+high CD48− CD34− CD229low/− and lymphoid-biased Vwf-tdTomato+ HSCs were LKS+ CD150+high CD48− CD34− CD229high [
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ].
      Note that Vwf-GFP and Vwf-tdTomato reporters reliably measure Vwf RNA; VWF protein was not confirmed to be expressed by the HSCs likely because it is intracellular and difficult to assess by flow cytometry methods. There is an excellent antibody against human VWF that crossreacts with mouse, and we have used it to detect VWF+ cells in paraffin-embedded BM sections obtained from both species [
      • Duarte D
      • Hawkins ED
      • Akinduro O
      • et al.
      Inhibition of endosteal vascular niche remodeling rescues hematopoietic stem cell loss in AML.
      ,
      • Tjin G
      • Flores-Figueroa E
      • Duarte D
      • et al.
      Imaging methods used to study mouse and human HSC niches: current and emerging technologies.
      ]. It would therefore be of interest to assess VWF expression on cytospins of Vwf-GFP/tdTomato+ HSCs by immunohistochemistry-based methods to conclusively determine whether all Vwf-GFP or Vwf-tdTomato+ HSCs do express VWF protein.

      CD41 MYELOID-BIASED HSCs

      In complementary studies to those using the Vwf-GFP/tdTomato HSCs, Gekas and Graf [
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      ] showed that CD41 is expressed on a population of adult murine HSCs (defined as either LKS+ CD34− CD135− or LKS+ CD150+ CD48−) and that CD41-expressing HSCs increased with aging, marking the majority of HSCs in 16-month-old C57BL/6 mice. CD41 had previously been shown to separate definitive hematopoietic cells (which are CD41+) from endothelial cells (which lack expression of CD41) in the mouse embryo [
      • Mikkola HK
      • Fujiwara Y
      • Schlaeger TM
      • Traver D
      • Orkin SH.
      Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo.
      ,
      • Ferkowicz MJ
      • Starr M
      • Xie X
      • et al.
      CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo.
      ], and CD41 has been shown to be expressed by yolk sac, fetal, placental, and a small proportion of adult HSCs [
      • Mikkola HK
      • Fujiwara Y
      • Schlaeger TM
      • Traver D
      • Orkin SH.
      Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo.
      ,
      • Ferkowicz MJ
      • Starr M
      • Xie X
      • et al.
      CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo.
      ,
      • Rhodes KE
      • Gekas C
      • Wang Y
      • et al.
      The emergence of hematopoietic stem cells is initiated in the placental vasculature in the absence of circulation.
      ,
      • Robin C
      • Ottersbach K
      • Boisset JC
      • Oziemlak A
      • Dzierzak E.
      CD41 is developmentally regulated and differentially expressed on mouse hematopoietic stem cells.
      ]. Interestingly, the studies by Sanjuan-Pla et al. [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ] identified that approximately 68% of Vwf-GFP+ HSCs coexpressed CD41 protein.
      Ferkowicz et al. [
      • Ferkowicz MJ
      • Starr M
      • Xie X
      • et al.
      CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo.
      ] had previously investigated the function of CD41+ adult HSCs and reported that HSC activity was enriched in the CD41−/lo population [
      • Ferkowicz MJ
      • Starr M
      • Xie X
      • et al.
      CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo.
      ]. This is consistent with the findings of the Morrison group, who reported that CD41+ cells did not reconstitute hematopoiesis when transplanted into mice [
      • Kiel MJ
      • Yilmaz OH
      • Iwashita T
      • Yilmaz OH
      • Terhorst C
      • Morrison SJ.
      SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.
      ]. However, Gekas and Graf made the important observation that the CD41 antibody, (clone MWReg30), is a blocking antibody [
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      ]. This meant that it could potentially interfere with the engraftment and, in turn, reconstitution of the CD41-expressing HSCs. To test this, they incubated BM cells obtained from WT mice with either MWReg30 or the control immunoglobulin G (IgG) antibody for 30 min, then mixed the cells 1:1 with untreated competing BM cells and injected them into lethally irradiated congenic mice [
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      ]. There was no difference in the proportions of donor-derived cells in the recipient mice at 1 month after transplantation (a time point that assesses the repopulation of hematopoiesis from MPPs). By contrast, at both 2 and 4 months after transplantation, the donor-derived repopulation was significantly reduced (by approximately 50%) in recipients that received the MWReg30-treated BM compared with those that received IgG control-treated BM. The MWReg30 clone was used in the two previous studies which concluded that adult HSCs were CD41−, thus resolving this discrepancy in the literature [
      • Kiel MJ
      • Yilmaz OH
      • Iwashita T
      • Yilmaz OH
      • Terhorst C
      • Morrison SJ.
      SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.
      ,
      • Ferkowicz MJ
      • Starr M
      • Xie X
      • et al.
      CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo.
      ]. It was also used in the studies by Carrelha et al. [
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ] in their search for cell surface markers that could be used to purify Vwf-tdTomato+ platelet-biased and lymphoid-biased HSCs. They reported that there were no differences in the repopulating activity of the CD41+ and CD41− Vwf-tdTomato+ HSCs [
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ]; however, the blocking effects of the CD41 antibody likely contributed to these results. Furthermore, in some single-HSC studies of latent HSCs and MyRPs, Yamamoto et al. [
      • Yamamoto R
      • Morita Y
      • Ooehara J
      • et al.
      Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.
      ,
      • Yamamoto R
      • Wilkinson AC
      • Ooehara J
      • et al.
      Large-scale clonal analysis resolves aging of the mouse hematopoietic stem cell compartment.
      ] used CD41 expression to isolate the cells before transplantation, and this may also have influenced the data. It is highly recommended that CD41 not be used to isolate HSCs for transplantation studies.
      To explore the role of CD41 in adult HSCs, Gekas and Graf used CD41YFP knock-in mice, which they had previously generated by inserting the Eyfp gene at the start site of the GpIIb (CD41) locus in embryonic stem cells [
      • Zhang J
      • Varas F
      • Stadtfeld M
      • Heck S
      • Faust N
      • Graf T.
      CD41-YFP mice allow in vivo labeling of megakaryocytic cells and reveal a subset of platelets hyperreactive to thrombin stimulation.
      ]. Homozygous CD41YFP/YFP mice were shown to be akin to CD41 knockout (KO) mice [
      • Zhang J
      • Varas F
      • Stadtfeld M
      • Heck S
      • Faust N
      • Graf T.
      CD41-YFP mice allow in vivo labeling of megakaryocytic cells and reveal a subset of platelets hyperreactive to thrombin stimulation.
      ] and were subsequently used (and termed CD41-KO) to determine whether CD41 had an important functional role in adult hematopoiesis. At two months of age, the CD41YFP/YFP (CD41-KO) mice exhibited peripheral blood pancytopenia in all lineages (platelets, erythrocytes myeloid cells, B lymphocytes, and T lymphocytes), and this worsened in 9 month- and 10 month-old-mice. The CD41-KO mice had a hypocellular BM at all the time points assessed.
      The numbers of HSCs (LKS+ CD34− CD135−) were significantly increased in the CD41-KO mice; however, this was accompanied by notable reductions in ST-HSC (LKS+ CD34+ CD135−) and MPPLy (LKS+ CD34+ CD135+). The LT-HSC and a mixed population comprising both ST-HSC and MPPLy (LKS+ CD34+) were shown to have increased apoptosis when compared with the same populations obtained from WT C57BL/6 mice. Furthermore, the CD41-KO LT-HSC, but not the LKS+CD34+ (ST-HSC/MPPLy) cells, had an increased proliferation rate, assessed at 16 hours after injection of bromodeoxyuridine (BrdU) into mice [
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      ]. Transplantation studies confirmed that the defects observed in CD41-KO mice were intrinsic to the HSC.
      To determine whether the CD41-KO HSCs had altered competitive repopulation capacity, 2 × 105 CD41-KO or WT CD45.2+ BM cells were transplanted together with 2 × 105 competing CD45.1+ BM cells into CD45.1/CD45.2 recipients and were assessed for donor cell contribution for up to 6 months after transplantation. The CD41-KO BM cells had an increased donor cell contribution at 1 month after transplantation, but similar donor cell reconstitution at 2 and 4 months after transplantation when compared with WT BM. However, the recipient mice of the CD41-KO BM competitive bone marrow transplant had thrombocytopenia and leukopenia at both 4 and 6 months after transplantation. Additional analyses revealed that the contribution of the WT competing cells to hematopoiesis had been altered when cotransplanted with the CD41-KO BM cells, revealing a feedback mechanism from CD41-KO BM cells that influenced WT hematopoiesis and contributed to the pancytopenia [
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      ]. Although the mechanism was not determined, it is likely that the depletion of megakaryocytes, which also express CD41+ and have been shown to have important roles in regulating HSCs [
      • Pinho S
      • Marchand T
      • Yang E
      • Wei Q
      • Nerlov C
      • Frenette PS
      Lineage-biased hematopoietic stem cells are regulated by distinct niches.
      ,
      • Bruns I
      • Lucas D
      • Pinho S
      • et al.
      Megakaryocytes regulate hematopoietic stem cell quiescence through CXCL4 secretion.
      ,
      • Nakamura-Ishizu A
      • Takubo K
      • Fujioka M
      • Suda T.
      Megakaryocytes are essential for HSC quiescence through the production of thrombopoietin.
      ,
      • Nakamura-Ishizu A
      • Takubo K
      • Kobayashi H
      • Suzuki-Inoue K
      • Suda T.
      CLEC-2 in megakaryocytes is critical for maintenance of hematopoietic stem cells in the bone marrow.
      ], contributed to this phenotype.
      Microarray studies using WT mice revealed that CD41+ LKS+ CD135− HSCs had increased expression of genes associated with megakaryocyte/platelets (including Vwf, Selp, and Pf4) and myeloerythroid and megakaryocyte transcription factors, including Gata1, Zfpm1, Gfi1b and Klf1. Furthermore, the CD41–LKS+ CD135− HSCs were enriched in lymphoid-restricted genes, including Ftl3, Il7ra, and members of the Ikaros and Notch families. The microarray studies also implicated that the CD41+ HSCs were more quiescent than the CD41− HSCs, and this was confirmed by BrdU-labeling studies in WT mice [
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      ].
      The numbers of CFCs formed from single-sorted cells were similar for both the CD41− and CD41+ HSCs obtained from WT mice; however, the CD41+ HSCs produced smaller CFCs and had reduced proliferative potential in liquid cultures. Studies performed in vitro indicated that, similar to what was observed for the Vwf-GFP+ HSCs, the CD41+ HSCs were able to form CD41− HSCs, whereas the CD41− HSCs had limited capacity to produce CD41+ HSCs, although a small, transient population of immunophenotypical CD41+ HSCs were produced from CD41− HSCs at early time points postculture initiation [
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      ].
      Finally, using heterozygous CD41YFP/+ mice (which were shown to have similar hematopoietic cell content to WT mice), they sorted YFP+ (CD41+) and YFP− (CD41−) LT-HSCs (defined as LKS+ CD150+ CD48−) and transplanted 50–100 LT-HSCs together with 2 × 105 competing CD45.1+ BM cells into lethally irradiated recipients. The YFP+ (CD41+) LT-HSCs had notably increased myeloid reconstitution accompanied by notably reduced B lymphocyte repopulation in primary recipients compared with the YFP− (CD41−) LT-HSCs. Although slightly increased in secondary recipients, the myeloid bias was not significantly different to that of the YFP− (CD41−) HSCs [
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      ]. Interestingly, however, most secondary recipients of the BM obtained from primary recipients of the YFP+ HSCs had high donor cell chimerism, whereas the donor cell chimerism observed in YFP− HSCs was variable [
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      ]. Unlike the studies by Sanjuan-Pla et al. [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ], the authors did not investigate whether the YFP− (CD41−) LT-HSCs could form YFP+ (CD41+) LT-HSCs and vice versa after transplantation, which would have been of interest. Intriguingly, in validation studies that used BM cells obtained from the CD41YFP/+ mice, all c-KIT+ YFP+ cells were shown to express CD41, whereas not all c-KIT+ CD41+ cells expressed YFP [
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      ]. The reasons for this inconsistency in coexpression was unclear. A summary of these studies is provided in Table 5.
      Table 5Properties of HSCs identified using CD41
      Original nameCD41+ HSC
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      CD41− HSC
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      CD41YFP/+ HSC
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      CD41YFP/−HSCs
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      Revised nameMyeloid-biased HSCLymphoid-biased HSCCD41+ HSCsCD41- HSCs
      Flow cytometry gating strategyLKS+ CD41+ CD34− CD135− or LKS+ CD41+ CD150+ CD48−

      LKS+ CD41− CD34− CD135− or LKS+ CD41− CD150+ CD48−

      CD41YFP+ LKS+ CD150+ CD48− LT-HSCsCD41YFP − LKS+ CD150+CD48− LT-HSCs
      Primary transplant

      Competitive repopulation potential when 50–100 cells were cotransplanted with 2 × 105 wild-type BM cells (repopulation assessed at 4 months, all mice >0.1% donor cells)
      ND (CD41 was identified to be a blocking antibody, all HSC transplants were performed using CD41YFP/+ mice)ND (CD41 was identified to be a blocking antibody, all HSC transplants were performed using CD41YFP/- mice)Robust repopulation (all 21 recipients had >1% donor cells).

      A myeloid bias was observed, however, all mice showed donor-derived repopulation in all lineages
      11/15 mice had >1% donor cells.

      A lymphoid-bias was observed, however, all mice showed donor-derived repopulation in all lineages
      Secondary transplant

      BM cells (1/10th of a femur) obtained from primary recipients of CD41YFP/+ HSCs
      NDNDAll 10 recipients had >10% donor-derived reconstitution.

      A myeloid bias was observed, however all lineages were repopulated with the exception of one mouse which lacked donor-derived repopulation in B lymphocytes.
      5/10 recipients had >10% donor-derived reconstitution.

      A lymphoid-bias was observed, however, all lineages were repopulated in 7/10 recipients
      Potential to form other HSC typeYes, assessed in liquid culture onlyTransient at early time points after culture initiation onlyNDND
      Cell cycle (assessed at 16 hours after injection of BrdU into mice)Approximately 2% BrdU+Approximately 10% BrdU+NDND
      CFC potential from single-sorted cells∼85% formed CFCs

      The majority formed small colonies (<40 cells)
      ∼85% formed CFCs

      The majority formed large colonies (>2mm in size)
      NDND
      BM = bone marrow; BrdU = bromodeoxyuridine; CFC = colony-forming cell; HSC = hematopoietic stem cell; LT-HSC = long-term repopulating hematopoietic stem cell; ND = not determined.

      EVIDENCE FOR CD41+ AND VWF+ HSCs IN HUMANS

      It was previously shown that CD41 was expressed on human cord blood CD34+ cells and, to a lesser extent, on adult G-CSF mobilized CD34+ peripheral blood cells [
      • Debili N
      • Robin C
      • Schiavon V
      • et al.
      Different expression of CD41 on human lymphoid and myeloid progenitors from adults and neonates.
      ]. Comparisons of the properties of CD34+ CD41+ CD42− cells with CD34+ CD41− cells by limiting dilution analyses of long-term culture-initiating cells (LTC-IC [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ]) indicated that the CD34+ CD41− cells had a higher frequency of LTC-IC [
      • Debili N
      • Robin C
      • Schiavon V
      • et al.
      Different expression of CD41 on human lymphoid and myeloid progenitors from adults and neonates.
      ]. Both populations obtained from cord blood were capable of multilineage repopulation in NOD/SCID mice, with 9 of 10 mice transplanted with CD34+ CD41− CD42− cells and 6 of 10 mice transplanted with CD34+ CD41+ CD42− cells being positive for human cells [
      • Debili N
      • Robin C
      • Schiavon V
      • et al.
      Different expression of CD41 on human lymphoid and myeloid progenitors from adults and neonates.
      ]. The cord blood CD34+ CD41− CD42− and CD34+ CD41+ CD42− cells had comparable potential to produce T lymphocytes in the in vitro NOD/SCID embryonic thymus hanging drop assay [
      • Debili N
      • Robin C
      • Schiavon V
      • et al.
      Different expression of CD41 on human lymphoid and myeloid progenitors from adults and neonates.
      ]. It is unclear whether, similar to what was observed for mice, the human CD41 antibody used in these studies (clone Tab) is a blocking antibody, which could affect the readout of the in vivo assays.
      More recently, single-cell analysis of human fetal and adult BM HSCs revealed that VWF was expressed by a subset of these HSCs [
      • Notta F
      • Zandi S
      • Takayama N
      • et al.
      Distinct routes of lineage development reshape the human blood hierarchy across ontogeny.
      ]. The function of the VWF-expressing HSC population is yet to be determined and would require the identification of cell surface markers that exclusively identify the VWF-expressing HSCs. Furthermore, it is not clear whether, as identified for mice [
      • Gekas C
      • Graf T.
      CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.
      ], the VWF-expressing HSCs coexpress, at least to some extent, CD41.

      UPDATES ON ASSAYS USED TO ASSESS HSCs AND MPPs: THEIR BENEFITS AND THEIR LIMITATIONS

      In our previous review [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ], we also discussed key assays that are used to assess the functions of HSCs and MPPs; this section provides a brief update on such assays.

      SUBLETHALLY IRRADIATED RECIPIENTS OF HSCs AND MPPs

      One of the in vivo assays from the Passegue laboratory assessed the repopulation potential of the HSC and MPP populations in sublethally irradiated recipients [
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ]. This assay enables transplantation of HSC and MPP populations without requiring support BM cells to keep the mice alive. These studies used β-actin–GFP mice as donors to enable assessment of the donor cells to platelet reconstitution (platelets do not express CD45.1 or CD45.2, which is the most commonly used method of detecting donor-derived cells in transplant studies in mice [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ]). The contribution of the GFP+ donor cells to platelets, nucleated cells, and CD11b+ myeloid cells was assessed in the peripheral blood at regular intervals between 7 and 34 days after transplantation. The populations were transplanted at two different cell doses: 2,000 or 500 cells, the latter reported for the LT-HSC, ST-HSC, and MPP2 populations only.
      These studies revealed that 2,000 LT-HSC, ST-HSC, and MPP2 cells robustly reconstituted platelets, nucleated cells, and myeloid cells for more than 1 month after transplantation. By contrast, 2,000 MPP3 and MPP4 cells had limited in vivo repopulating potential, with less than 20% donor contribution to platelets and nucleated cells. The MPP3 showed a transient myeloid bias that notably declined by day 34 after transplantation, whereas MPP4 produced fewer myeloid cells after the first 14 days after transplantation. When 500 cells were transplanted, the LT-HSC had superior platelet repopulating capacity compared with that of ST-HSC and MPP2 cells; however, myeloid repopulation was similar for LT-HSC and ST-HSC.
      There were two caveats of this study. First, the individual mice were bled multiple times (every 3–4 days) between 7 and 34 days after transplantation to monitor blood cell repopulation. Other investigators have shown that repeated blood sampling can cause hematopoietic stress and alter responses of HSCs (and MPPs), primarily in response to anemia that can occur in response to regular bleeding [
      • Cheshier SH
      • Prohaska SS
      • Weissman IL.
      The effect of bleeding on hematopoietic stem cell cycling and self-renewal.
      ,
      • Boggs SS
      • Boggs DR.
      Effect of bleeding on hematopoiesis following irradiation and marrow transplantation.
      ,
      • Inra CN
      • Zhou BO
      • Acar M
      • et al.
      A perisinusoidal niche for extramedullary haematopoiesis in the spleen.
      ]. Although these prior studies removed high volumes of blood in each bleed (250 µL and above), it has been recommended that mice be bled a maximum of 150 µL/25 g mouse per week and no more than 200 µL/25 g mouse during two weeks [

      How much blood can I take from a mouse without endangering its health? The Jackson Laboratory. October 1, 2005. Available at:https://www.jax.org/news-and-insights/2005/october/how-much-blood-can-i-take-from-a-mouse-without-endangering-its-health. Accessed October 30, 2022.

      ]. This recommendation has been based on weekly bleeds of larger volumes, not repeated bleeds within the duration of a week; however, it is important to consider the effects of repeated blood sampling on mice, and it has been recommended that the effects of repeated bleeding on erythrocyte parameters (hemoglobin and hematocrit) be monitored during such experiments [

      How much blood can I take from a mouse without endangering its health? The Jackson Laboratory. October 1, 2005. Available at:https://www.jax.org/news-and-insights/2005/october/how-much-blood-can-i-take-from-a-mouse-without-endangering-its-health. Accessed October 30, 2022.

      ]. Pietras et al. [
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ] did not report erythroid parameters in their studies.
      Second, the repopulation of the mice was assessed for less than 40 days, whereas it is essential to monitor repopulation for a minimum of 16 weeks after transplantation to assess HSCs [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ,
      • Dykstra B
      • Kent D
      • Bowie M
      • et al.
      Long-term propagation of distinct hematopoietic differentiation programs in vivo.
      ]. It is unclear why they did not monitor the mice beyond 40 days; however, C57BL/6 mice that are irradiated can be susceptible to developing radiation-induced lymphoma, including sublethally irradiated recipients (extensively reviewed by Rivina et al [
      • Rivina L
      • Davoren MJ
      • Schiestl RH.
      Mouse models for radiation-induced cancers.
      ] and Purton laboratory, unpublished observations).

      THE CHOICE OF COMPETING CELLS CAN INFLUENCE THE READOUT OF COMPETITIVE REPOPULATING IN VIVO ASSAYS

      It has previously been recognized that unfractionated BM cells obtained from mice, (which are used as a standard source of competing BM cells in both competitive repopulation assays [
      • Harrison DE.
      Competitive repopulation: a new assay for long-term stem cell functional capacity.
      ] and limiting dilution assays [
      • Szilvassy SJ
      • Humphries RK
      • Lansdorp PM
      • Eaves AC
      • Eaves CJ.
      Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy.
      ], both of which are explained in our previous review [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ]) contain long-lived B and T lymphocytes [
      • Rundberg Nilsson A
      • Pronk CJ
      • Bryder D
      Probing hematopoietic stem cell function using serial transplantation: seeding characteristics and the impact of stem cell purification.
      ]. However, there are some recent studies that have highlighted additional issues that researchers need to be aware of when using whole BM for such experiments. First, studies from the Bryder laboratory identified that unfractionated BM also contains long-lived progenitor cells, and these can influence the readout of secondary transplantations [
      • Rundberg Nilsson A
      • Pronk CJ
      • Bryder D
      Probing hematopoietic stem cell function using serial transplantation: seeding characteristics and the impact of stem cell purification.
      ]. This study also showed that the distribution of HSCs in bones obtained from the same mouse can be very different. To overcome this, it is recommended to pool bones from the same mouse before undertaking serial transplant studies.
      Carrelha et al. [
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ] also made some very interesting observations about the lineage reconstitution of the single HSCs when competed against either WT or W41/W41 BM [
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ]. Although similar proportions of recipients with distinct lineage biases were observed when either competing BM source was used, the use of WT BM notably increased the detection of lymphoid-biased HSCs and HSCs that repopulated multilineages equivalently. By contrast, the use of W41/W41 BM significantly increased the detection of platelet/erythroid-biased HSCs and platelet/erythroid/myeloid-biased HSCs [
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ]. It is likely that this occurred because c-KIT-expressing hematopoietic cells in W41/W41 mice have an impaired response to c-KIT ligand, which is essential in regulating many of the erythroid and myeloid lineages in addition to megakaryocyte progenitors [
      • Pronk CJH
      • Rossi DJ
      • Månsson R
      • et al.
      Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy.
      ].

      TIME AFTER TRANSPLANTATION AT WHICH REPOPULATING POTENTIAL IS ASSESSED

      A caveat of most studies reviewed here is that although 16 weeks after transplantation is considered sufficient to assess HSCs [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ], this may be too early to conclusively differentiate LT-HSCs from ST-HSCs. Transplantation studies by Benenviste et al. [
      • Benveniste P
      • Frelin C
      • Janmohamed S
      • et al.
      Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential.
      ] suggested that there is an intermediate HSC (superior to ST-HSCs) that can repopulate mice at the clonal level for 6–8 months after transplantation. This study is consistent with a seminal study, in which Jordan and Lemischka [
      • Jordan CT
      • Lemischka IR
      Clonal and systemic analysis of long-term hematopoiesis in the mouse.
      ] used integration site analyses of oncoretrovirally transduced HSCs to show that multiple clones give rise to hematopoiesis between 4 and 6 months after transplantation, after which stable clonal hematopoiesis will occur. The findings of these two independent studies are very important to consider, and I recommend that a minimum of 26 weeks after transplantation should be used to adequately assess HSCs.

      RECENT ADVANCES IN TECHNOLOGIES THAT HAVE IMPROVED OUR UNDERSTANDING OF HSCs

      It is important to briefly highlight here that technological advances in the past few years have made notable contributions to our ability to identify and study HSCs and MPPs. The first two tools discussed below are innovative methods that have advanced single-cell studies of HSCs. A recent elegant review by Rodriguez-Fraticelli and Camargo [
      • Rodriguez-Fraticelli AE
      • Camargo F.
      Systems analysis of hematopoiesis using single-cell lineage tracing.
      ] thoroughly discussed these two types of single-cell technologies, including caveats of each type of approach, and the pioneering studies are briefly highlighted here.
      Cellular barcoding studies have enabled the ability of investigators to track HSCs and their progeny. These studies have included different approaches including gene transduction [
      • Gerrits A
      • Dykstra B
      • Kalmykowa OJ
      • et al.
      Cellular barcoding tool for clonal analysis in the hematopoietic system.
      ], transposons [
      • Sun J
      • Ramos A
      • Chapman B
      • et al.
      Clonal dynamics of native haematopoiesis.
      ], CRISPR-Cas9 technology [
      • Bowling S
      • Sritharan D
      • Osorio FG
      • et al.
      An engineered CRISPR-Cas9 mouse line for simultaneous readout of lineage histories and gene expression profiles in single cells.
      ], and Polylox barcoding [
      • Pei W
      • Feyerabend TB
      • Rössler J
      • et al.
      Polylox barcoding reveals haematopoietic stem cell fates realized in vivo.
      ] of HSCs. Seminal contributions from the laboratories who have pioneered the use of cellular barcoding in HSC studies are highlighted here. These are collaborative studies from the de Haan and Bystrykh laboratories, which have used retroviral gene transduction methods of cellular barcoding methods to study the behavior of murine HSCs, including during leukemia development [
      • Gerrits A
      • Dykstra B
      • Kalmykowa OJ
      • et al.
      Cellular barcoding tool for clonal analysis in the hematopoietic system.
      ,
      • Klauke K
      • Broekhuis MJC
      • Weersing E
      • et al.
      Tracing dynamics and clonal heterogeneity of Cbx7-induced leukemic stem cells by cellular barcoding.
      ] and monitoring human HSCs in murine xenotransplantation studies [
      • Belderbos ME
      • Jacobs S
      • Koster TK
      • et al.
      Donor-to-donor heterogeneity in the clonal dynamics of transplanted humancord blood stem cellsin murine xenografts.
      ,
      • Jacobs S
      • Ausema A
      • Zwart E
      • et al.
      Detection of chemotherapy-resistant patient-derived acute lymphoblastic leukemia clones in murine xenografts using cellular barcodes.
      ,
      • Jacobs S
      • Ausema A
      • Zwart E
      • et al.
      Quantitative distribution of patient-derived leukemia clones in murine xenografts revealed by cellular barcodes.
      ]. The Camargo laboratory contributed Sleeping Beauty transposon [
      • Sun J
      • Ramos A
      • Chapman B
      • et al.
      Clonal dynamics of native haematopoiesis.
      ,
      • Rodriguez-Fraticelli AE
      • Wolock SL
      • Weinreb CS
      • et al.
      Clonal analysis of lineage fate in native haematopoiesis.
      ], lentiviral [
      • Rodriguez-Fraticelli AE
      • Weinreb C
      • Wang SW
      • et al.
      Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis.
      ], and CRISPR-Cas9 [
      • Bowling S
      • Sritharan D
      • Osorio FG
      • et al.
      An engineered CRISPR-Cas9 mouse line for simultaneous readout of lineage histories and gene expression profiles in single cells.
      ] barcoding approaches to study murine HSCs, including in native and stressed conditions. The Rodewald laboratory developed the Polylox method, generating a number of useful Cre-loxP-driven barcoding approaches including Rosa26Polylox/+ C57BL/6 mice [
      • Pei W
      • Feyerabend TB
      • Rössler J
      • et al.
      Polylox barcoding reveals haematopoietic stem cell fates realized in vivo.
      ,
      • Pei W
      • Shang F
      • Wang X
      • et al.
      Resolving fates and single-cell transcriptomes of hematopoietic stem cell clones by PolyloxExpress barcoding.
      ,
      • Pei W
      • Wang X
      • Rössler J
      • Feyerabend TB
      • Höfer T
      • Rodewald HR
      Using cre-recombinase-driven Polylox barcoding for in vivo fate mapping in mice.
      ]. All these approaches enabled the tracking of HSCs and their progeny, although the sensitivity of identifying HSCs in each of the distinct approaches is different [
      • Rodriguez-Fraticelli AE
      • Camargo F.
      Systems analysis of hematopoiesis using single-cell lineage tracing.
      ].
      The development of single-cell RNA-sequencing (scRNA-seq) has advanced the ability to assess gene expression in HSCs at the single-cell level. Notable scRNA-seq studies of murine HSCs that have pioneered studies focused on resolving the heterogeneity of the immunophenotypic definition of healthy murine HSCs are collaborative studies from the Göttgens and Kent laboratories [
      • Wilson NK
      • Kent DG
      • Buettner F
      • et al.
      Combined single-cell functional and gene expression analysis resolves heterogeneity within stem cell populations.
      ,
      • Che JLC
      • Bode D
      • Kucinski I
      • et al.
      Identification and characterization of in vitro expanded hematopoietic stem cells.
      ]. These studies have collectively developed gene sets to identify different types of HSCs and MPPs that are valuable resources for the HSC community.
      In addition to the Cre-driven mouse strains that target HSCs and can be crossed to reporter strains for tracing studies (a number previously reviewed by Joseph et al [
      • Joseph C
      • Quach JM
      • Walkley CR
      • Lane SW
      • Lo Celso C
      • Purton LE
      Deciphering hematopoietic stem cells in their niches: a critical appraisal of genetic models, lineage tracing, and imaging strategies.
      ]) and the Vwf-GFP [
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ] and Vwf-tdTomato [
      • Carrelha J
      • Meng Y
      • Kettyle LM
      • et al.
      Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.
      ] reporter mice discussed above, some additional HSC reporter mice have recently been generated to study HSCs. These reporter mice include Evi1-IRES-GFP [
      • Kataoka K
      • Sato T
      • Yoshimi A
      • et al.
      Evi1 is essential for hematopoietic stem cell self-renewal, and its expression marks hematopoietic cells with long-term multilineage repopulating activity.
      ], Fgd5-ZSGreen [
      • Gazit R
      • Mandal PK
      • Ebina W
      • et al.
      Fgd5 identifies hematopoietic stem cells in the murine bone marrow.
      ], Hoxb5-tri-mCherry [
      • Chen JY
      • Miyanishi M
      • Wang SK
      • et al.
      Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche.
      ], Pdzk1ip1-GFP [
      • Sawai CM
      • Babovic S
      • Upadhaya S
      • et al.
      Hematopoietic stem cells are the major source of multilineage hematopoiesis in adult animals.
      ], Krt18 Cre-ER mice crossed to either EYFP or tdTomato reporter strains [
      • Chapple RH
      • Tseng YJ
      • Hu T
      • et al.
      Lineage tracing of murine adult hematopoietic stem cells reveals active contribution to steady-state hematopoiesis.
      ], Krt7-GFP [
      • Tajima Y
      • Ito K
      • Umino A
      • Wilkinson AC
      • Nakauchi H
      • Yamazaki S.
      Continuous cell supply from Krt7-expressing hematopoietic stem cells during native hematopoiesis revealed by targeted in vivo gene transfer method.
      ], Gprc5c-EGFP [
      • Cabezas-Wallscheid N
      • Buettner F
      • Sommerkamp P
      • et al.
      Vitamin A-retinoic acid signaling regulates hematopoietic stem cell dormancy.
      ], and Hlf-tdTomato [
      • Yokomizo T
      • Watanabe N
      • Umemoto T
      • et al.
      Hlf marks the developmental pathway for hematopoietic stem cells but not for erythro-myeloid progenitors.
      ].
      Combinations of the aformentioned innovative technologies together with immunophenotypical markers and assays of HSCs can be used to further understand HSCs at the single-cell level, as recently reviewed by Rodriguez-Fraticelli and Camargo [
      • Rodriguez-Fraticelli AE
      • Camargo F.
      Systems analysis of hematopoiesis using single-cell lineage tracing.
      ].

      QUESTIONS TO BE RESOLVED IN FUTURE STUDIES

      It is an exciting time in the field of HSC biology, with many questions yet to be resolved. First, can additional markers (e.g., combining the cell surface markers used by different investigators) be used to further define the HSC hierarchy? Indeed, in studies from the Pietras laboratory EPCR was recently combined with LKS+, CD135, CD48, and CD150 to reveal that EPCR can be used to further purify four populations of SLAM HSCs (LKS+, CD135− CD48− CD150+) that were subdivided on the basis of their expression (or lack of expression) of CD34 and/or EPCR [
      • Rabe JL
      • Hernandez G
      • Chavez JS
      • Mills TS
      • Nerlov C
      • Pietras EM.
      CD34 and EPCR coordinately enrich functional murine hematopoietic stem cells under normal and inflammatory conditions.
      ]. They showed that the CD34-EPCR+ SLAM HSCs had the most robust repopulating potential and were less susceptible to the effects of chronic interleukin 1 (IL-1) infection [
      • Rabe JL
      • Hernandez G
      • Chavez JS
      • Mills TS
      • Nerlov C
      • Pietras EM.
      CD34 and EPCR coordinately enrich functional murine hematopoietic stem cells under normal and inflammatory conditions.
      ]. These studies also revealed that Vwf-GFP-expressing cells were notably reduced in LKS+ CD135− CD48− CD150+ HSCs after chronic IL-1 exposure. By contrast, the expression of Fgd5-ZSGreen-positive cells in the SLAM HSCs was unaltered after IL-1 exposure [
      • Rabe JL
      • Hernandez G
      • Chavez JS
      • Mills TS
      • Nerlov C
      • Pietras EM.
      CD34 and EPCR coordinately enrich functional murine hematopoietic stem cells under normal and inflammatory conditions.
      ]. Fgd5-ZSGreen is another reporter mouse that has been used to identify HSCs in mice and, intriguingly, has been shown to be essential for embryonic, but not definitive hematopoiesis [
      • Gazit R
      • Mandal PK
      • Ebina W
      • et al.
      Fgd5 identifies hematopoietic stem cells in the murine bone marrow.
      ].
      Furthermore, with the exception of the studies from the Eaves laboratory [
      • Dykstra B
      • Kent D
      • Bowie M
      • et al.
      Long-term propagation of distinct hematopoietic differentiation programs in vivo.
      ,
      • Kent DG
      • Copley MR
      • Benz C
      • et al.
      Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential.
      ], all other aforementioned flow cytometry gating strategies used Sca-1 positive cells to isolate HSCs, whereas Sca-1 knockout mice are viable and are generated at normal Mendelian frequencies [
      • Ito CY
      • Li CY
      • Bernstein A
      • Dick JE
      • Stanford WL.
      Hematopoietic stem cell and progenitor defects in Sca-1/Ly-6A-null mice.
      ]. Adult Sca-1 knockout mice do, however, have defective hematopoiesis. Intriguingly, these defects are reflective of reduced platelet/myeloid-biased HSCs, with the Sca-1 knockout mice having thrombocytopenia, increased lymphocytes, and reduced myeloid cells, accompanied by reduced numbers of myeloid progenitors and primary and secondary repopulating HSCs [
      • Ito CY
      • Li CY
      • Bernstein A
      • Dick JE
      • Stanford WL.
      Hematopoietic stem cell and progenitor defects in Sca-1/Ly-6A-null mice.
      ].
      This raises the second question. Have some of the recent HSC studies discussed earlier in this review identified distinct populations of HSCs and, if so, where and when do they arise? Numerous studies have previously reported that embryonic HSCs and adult HSCs are distinct and that different transcription factors are key regulators of such HSCs. For example, Scl/Tal1 has been shown to be essential for embryonic HSCs but not adult HSCs [
      • Mikkola HK
      • Klintman J
      • Yang H
      • et al.
      Haematopoietic stem cells retain long-term repopulating activity and multipotency in the absence of stem-cell leukaemia SCL/tal-1 gene.
      ]. Intriguingly, however, embryonic-derived Scl/Tal1-targeted HSCs do persist for long term in adult mice [
      • Göthert JR
      • Gustin SE
      • Hall MA
      • et al.
      In vivo fate-tracing studies using the Scl stem cell enhancer: embryonic hematopoietic stem cells significantly contribute to adult hematopoiesis.
      ], and the hSclCreERT transgenic strain has been used to reliably modify gene expression in adult HSCs by postnatal administration of tamoxifen [
      • Smeets MF
      • Tan SY
      • Xu JJ
      • et al.
      Srsf2P95H initiates myeloid bias and myelodysplastic/myeloproliferative syndrome from hemopoietic stem cells.
      ] (and Purton laboratory, unpublished observations). Intriguingly, EPCR, Vwf, and CD41 are all highly expressed by embryonic HSCs [
      • Kent DG
      • Copley MR
      • Benz C
      • et al.
      Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential.
      ,
      • Sanjuan-Pla A
      • Macaulay IC
      • Jensen CT
      • et al.
      Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.
      ,
      • Mikkola HK
      • Fujiwara Y
      • Schlaeger TM
      • Traver D
      • Orkin SH.
      Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo.
      ,
      • Ferkowicz MJ
      • Starr M
      • Xie X
      • et al.
      CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo.
      ,
      • Rhodes KE
      • Gekas C
      • Wang Y
      • et al.
      The emergence of hematopoietic stem cells is initiated in the placental vasculature in the absence of circulation.
      ,
      • Robin C
      • Ottersbach K
      • Boisset JC
      • Oziemlak A
      • Dzierzak E.
      CD41 is developmentally regulated and differentially expressed on mouse hematopoietic stem cells.
      ]. Future studies that resolve these questions will not only significantly contribute to our understanding of HSCs during health and disease but also potentially resolve the long-standing controversies on where HSCs reside and are regulated in adult mice [
      • Pinho S
      • Frenette PS.
      Haematopoietic stem cell activity and interactions with the niche.
      ].

      Conflict of Interest Disclosure

      The author declares that there is no conflict of interest regarding the publication of this article.

      Acknowledgments

      This review was supported, in part, by the Operational Infrastructure Support Program from the Victorian Government (to St. Vincent's Institute of Medical Research). Professor LE Purton is the recipient of the 2022 McCulloch and Till Award, bestowed by the International Society for Experimental Hematology (ISEH). We thank Mr. Wenxu Zhu and the reviewer for their helpful comments. This review is dedicated to Professor Connie Eaves in recognition of her numerous significant contributions to hematopoietic stem cell research, ISEH, and Experimental Hematology.

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