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Keywords:

  • Adult hematopoietic stem cells;
  • Cell adhesion molecules;
  • Long-term repopulation;
  • Self-renewal;
  • Hematopoietic stem cell transplantation;
  • Hematopoiesis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTERESTS
  10. REFERENCES
  11. Supporting Information

Hematopoietic stem cells (HSCs) reside in a specialized bone marrow (BM) microenvironment that supports the maintenance and functional integrity of long-term (LT)-HSCs throughout postnatal life. The objective of this work is to study the role of activated leukocyte cell adhesion molecule (Alcam) in HSC differentiation and self-renewal using an Alcam-null (Alcam−/−) mouse model. We show here that Alcam is differentially regulated in adult hematopoiesis and is highly expressed in LT-HSCs where its level progressively increases with age. Young adult Alcam−/− mice had normal homeostatic hematopoiesis and normal numbers of phenotypic HSCs. However, Alcam−/− HSCs had reduced long-term replating capacity in vitro and reduced long-term engraftment potential upon transplantation. We show that Alcam−/− BM contain a markedly lower frequency of long-term repopulating cells than wild type. Further, the long-term repopulating potential and engraftment efficiency of Alcam−/− LT-HSCs was greatly compromised despite a progressive increase in phenotypic LT-HSC numbers during long-term serial transplantation. In addition, an age-associated increase in phenotypic LT-HSC cellularity was observed in Alcam−/− mice. This increase was predominately within the CD150hi fraction and was accompanied by significantly reduced leukocyte output. Consistent with an aging-like phenotype, older Alcam−/− LT-HSCs display myeloid-biased repopulation activity upon transplantation. Finally, Alcam−/− LT-HSCs display premature elevation of age-associated gene expression, including Selp, Clu, Cdc42, and Foxo3. Together, this study indicates that Alcam regulates functional integrity and self-renewal of LT-HSCs. STEM CELLS2013;31:560–571


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTERESTS
  10. REFERENCES
  11. Supporting Information

Hematopoietic stem cells (HSCs) are capable of long-term self-renewal and multipotent differentiation to sustain life-long production of mature blood cells of all lineages. Adult long-term repopulating HSCs reside in specialized bone marrow (BM) niches along the endosteum and in perivascular sites adjacent to the endothelium [1–3]. The cell fate decisions of HSCs are controlled by cell intrinsic determinants, as well as cell extrinsic cues provided by the specialized niches. The intricate balance between HSC dormancy and activation, self-renewal and differentiation is critical in maintaining the functional integrity of HSCs throughout postnatal life. As with most tissues, aging of the hematopoietic system is associated with many clinically significant conditions, including diminution of the adaptive immune system, increased incidence of myeloid malignancies, and greater propensity for anemia [4–7]. Consistent with these clinical manifestations, functional studies of HSCs from older mice indicate a decline in stem cell capacity, attenuated lymphoid lineage output, and increased myeloid potential with age [8, 9]. These age-related alterations of HSCs, however, are associated with an increased frequency of cells that meet the most stringent phenotypic definitions of the HSC population in murine and human BM [10–13]. Furthermore, cell intrinsic alteration, such as skewing in lineage-associated gene expression and epigenetic remodeling, is associated with HSC aging [9, 11]. Additional microenvironmental mechanisms that regulate HSC self-renewal and lineage specification may also contribute to aging of the hematopoietic system.

Activated leukocyte cell adhesion molecule (ALCAM or CD166) is a cell surface immunoglobulin superfamily member that mediates homophilic adhesion and heterotypic interactions with CD6 [14, 15]. Alcam reportedly functions in biological processes as diverse as axon pathfinding and fasciculation [16, 17], vascular angiogenesis [18], migration of neurons and activated monocytes [19], and trafficking of leukocytes through the blood-brain barrier into the central nervous system [20]. Aberrant ALCAM expression has been associated with many cancer types with variable prognostic values [21–26]. Notably, ALCAM has been reported to mark a tumor-initiating cell population in prostate cancer and in lung cancer [27, 28]. In the hematopoietic system, ALCAM is expressed on activated lymphocytes and monocytes and on primitive human HSCs [29, 30]. In addition, Alcam is reportedly required for myeloid colony formation in avian embryonic hematopoietic progenitors [31], and Alcam-expressing endothelial cells can support murine embryonic hematopoiesis [18]. Engagement of ALCAM and CD6 is essential for dendritic cell-mediated T-cell stimulation [32], and ALCAM gene silencing was recently shown to be required for megakaryocytic differentiation [33]. Another recent study showed that an Alcam+/Sca1 subset of the endosteal niche cell population robustly supports HSC activity and exhibits differential expression of cell adhesion-related genes, suggesting that these cells regulate HSC function through cell adhesion [34]. Moreover, earlier studies of murine HSC aging suggested that Alcam expression is upregulated several fold in aged HSCs compared to young HSCs [9, 11]. Based on these observations, we hypothesized that Alcam might regulate adult HSC function related to age.

In the study described herein, we comprehensively investigated the role of Alcam in adult hematopoiesis and HSC function using an Alcam-null mouse model. A series of phenotypic and functional studies reveal that Alcam is important in ensuring long-term engraftment and the self-renewal of long-term HSCs (LT-HSCs) during transplantation and aging.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTERESTS
  10. REFERENCES
  11. Supporting Information

Mice

Alcam-null allele [17] backcrossed to C57Bl6 for more than eight generations was a generous gift from Joshua Weiner (University of Iowa). All mice were genotyped by polymerase chain reaction (PCR) using primers listed in supporting information Table 1. Two-month-old (young) or 12–16-month-old mice were used for experiments. All mice were maintained in an AAALAC-accredited animal facility at City of Hope, and all experimental procedures involving mice were performed in accordance with federal and state government guidelines and established institutional guidelines and protocols approved by the Institutional Animal Care and Use Committee at Beckman Research Institute of City of Hope.

Flow Cytometry and Cell Sorting

Femurs and tibias were crushed gently with a mortar and pestle to dissociate the BM fraction. Cells were resuspended in 5 mL phosphate buffered saline (PBS)/0.5% bovine serum albumin (BSA) and filtered through a 70-μm filter (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com). The following antibodies were used for fluorescence activated cell-sorting (FACS) analyses: CD117 (ckit, clone 2B8, eBioscience, San Diego, CA, http://us.ebioscience.com), Ly-6A/E (Sca-1, clone E13-161.7, BioLegend), CD150 (SLAM, clone TC15-12F12.2, BioLegend, San Diego, CA, http://www.biolegend.com), CD48 (clone HM48.1, BioLegend), CD34 (clone RAM34, eBioscience), CD16/CD32 (clone 93, eBioscience), CD127 (IL7R, clone A7R34, eBioscience), CD135 (Flk-2, Flt-3, Ly-72, clone A2F10, eBioscience), CD45.1 (clone A20, BioLegend), CD45.2 (clone 104, BioLegend), CD11b (clone M1/70, eBioscience), Ter119 (clone TER-119, BioLegend), CD45R (clone RA3-6B2, BD Biosciences), CD3 (clone 17A2, eBioscience), Ly-6G/Ly-6C (Gr1, clone RB6-8C5, BioLegend), and goat polyclonal anti-mouse ALCAM (CD166, R&D System, Minneapolis, MN, http://www.rndsystems.com). The lineage antibody cocktail included the following biotin-conjugated anti-mouse antibodies: CD19 (clone eBio1D3), NK-1.1 (clone PK136), CD45R (clone RA3-6B2), IgM (clone II/41), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8 (clone 53-6.7), Gr1 (clone RB6-8C5), CD127 (clone A7R34) at 1 μg/mL, CD11b (clone M1/70) at 2 μg/mL, and Ter119 (clone Ter119, from BioLegend) at 3 μg/mL. Secondary reagents used included streptavidin-PerCP-Cy5.5 (BioLegend), phycoerythrin (PE)-TexasRed (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), or V500 (BD Biosciences). Flow cytometry was performed on a four-laser, 14-detector FACS-LSRII (BD Biosciences). For cell sorting, lineage negative cells were enriched using EasySep lineage depletion reagents (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) according to the manufacturer's instructions. Phenotypic populations were defined as LT-HSCs (Lin/ckithi/Sca1+/Flt3/CD150+/CD48), short-term HSCs (ST-HSCs) (Lin/ckithi/Sca1+/Flt3/CD150/CD48), multipotent progenitors (MPPs) (Lin/ckithi/Sca1+/CD150+/−/CD48+), lymphoid-primed multipotent progenitors (LMPPs) (Lin/ckithi/Sca1+/Flt3+/CD150+/−/CD48+), common lymphoid progenitors (CLPs) (Lin/IL7R+/ckitlo/Sca1lo), myeloid progenitors (MPs) (Lin/ckit+/Sca1), common myeloid progenitors (CMPs) (Lin/ckit+/Sca1/CD34+/FcgRlo), granulocyte-macrophage progenitors (GMPs) (Lin/ckit+/Sca1/CD34+/FcgRhi), and megakaryocyte-erythroid progenitors (MEPs) (Lin/ckit+/Sca1/CD34/FcgRlo). Cell sorting was performed on a four-laser, 15-detector FACSAria-III or a six-laser, 18-detector FACSAria II SORP (BD Bioscience).

Transplantation Experiments

For competitive repopulation experiments, 2 × 105 unfractionated BM mononuclear cells isolated from Alcam−/− mice or WT littermates (CD45.2+) were transplanted intravenously into lethally irradiated (13 Gy) 6–8-week-old congenic C57BL/6 mice (CD45.1+/CD45.2+) together with 2 × 105 CD45.1+ unfractionated BM cells. Secondary transplantation was performed similarly using sorted CD45.2+ HSCs isolated from primary recipients 16 weeks after transplantation. Limiting dilution transplantation was similarly performed with three donor cell doses (2 × 105, 4 × 104, 8 × 103). For LT-HSC engraftment, 50 purified LT-HSCs from Alcam−/− mice or WT littermates (CD45.2+) were transplanted into lethally irradiated (13 Gy) 6–8-week-old CD45.1+ mice together with 2 × 105 CD45.1+ supportive cells. Engraftment of CD45.2+ cells was analyzed over 6 months and transplantation was repeated with 100 purified CD45.2+ LT-HSCs.

Quantitative Reverse Transcription PCR Analysis

RNA was isolated from sorted BM cells by using the RNeasy micro kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) according to the manufacturer's protocol. First-strand cDNA was generated using 200 U SuperScript III reverse transcriptase (Invitrogen) and 0.5 μg oligo dT primer in a 20 μL reaction. Quantitative reverse transcription PCR (qRT-PCR) was performed using LightCycler 480 SYBR Green I master mix (Roche Applied Science, Basel, Switzerland, http://www.roche-applied-science.com) containing 0.2 μM gene-specific primers and detected with a LightCycler 480 real-time PCR system (Roche Applied Science). Primers used are listed in supporting information Table 1, and relative expression levels were determined by the standard curve method. Alternative method using the TaqMan assay is described in supporting information Materials and Methods.

Statistics

Statistical analyses were performed with Student's t test or ANOVA for normal distribution. Mann–Whitney U tests were performed when normal distribution was not satisfied. p value less than 0.05 was considered statistically significant (*, p < .05; **, p < .01; ***, p < .001). Frequency estimation of limiting-dilution analysis was performed based on Poisson distribution using L-Calc (Stem Cell Technologies).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTERESTS
  10. REFERENCES
  11. Supporting Information

Alcam Is Highly Expressed in LT-HSCs and Is Progressively Upregulated with Age

As a first step toward understanding the function of Alcam in hematopoiesis, we assessed whether Alcam surface expression is differentially regulated in various phenotypically defined subsets of adult hematopoietic stem and progenitor cells (HSPCs) by immunostaining and flow cytometry (Fig. 1A). First, we analyzed young (2-month old) mice and found that Alcam was abundantly expressed in greater than 95% of primitive HSPCs, including phenotypically defined LT-HSCs, ST-HSCs, MPPs, and LMPPs (Fig. 1B, 1C). Alcam expression was differentially regulated among MP subsets and CLPs (Fig. 1B, 1C). Overall, GMPs expressed high levels of Alcam, while MEPs did not express detectable levels, and CMPs expressed intermediate levels. The CMP compartment could be divided into two subsets (Alcam+ and Alcam) based on Alcam expression (Fig. 1B, top). Similar differential Alcam surface expression was observed in HSPC subsets of 12-month-old mice (Fig. 1C). Interestingly, Alcam levels on the cell surface were significantly (p = .0159) elevated in 12-month-old LT-HSCs compared to those of 2-month-old (Fig. 1C). To determine whether Alcam expression is transcriptionally regulated, we analyzed Alcam mRNA levels in sorted LT-HSCs, ST-HSCs, MPPs, CMPs, MEPs, and GMPs by qRT-PCR and found a similar differential expression pattern as that observed with cell surface staining (Fig. 1D). These results indicate that Alcam is differentially regulated at the transcriptional level and is most highly expressed in the LT-HSC compartment. We also analyzed Alcam mRNA levels in HSPC subsets from young (2-month old), 12-month old, and 16-month old mice by qRT-PCR. Similar preferential expression in LT-HSCs is observed in all age groups, and we find a significant (p < .0001) age-associated upregulation of Alcam expression in LT-HSCs (Fig. 1E). An approximately twofold and fivefold increase in Alcam levels was detected at 12 months and 16 months, respectively.

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Figure 1. Alcam is highly expressed in primitive HSCs and is progressively upregulated with age. (A): Representative fluorescence-activated cell sorting profile illustrating gating strategies for hematopoietic stem and progenitor cell (HSPC) subsets. Gray arrows indicate further separation of the gated population. (B): Representative histograms of Alcam staining in MP (top) (CMP: dotted line; GMP: black shading; MEP: black solid line), LMPP (middle), and Lin/ckithi/Sca1+ (LSK) subsets (bottom) (LT-HSC: black shading; ST-HSC: dotted line; MPP: black solid line) or isotype control (gray shading). (C): Average Alcam median fluoresence intensity ± SEM (2-month-old: n = 5; 12-month-old: n = 4) determined by FACS. (D): Average relative Alcam mRNA levels in sorted populations, as detected by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR; n = 3, each performed in duplicate). (E): Relative Alcam mRNA levels in sorted populations from 2-month-, 12-month-, and 16-month-old wild type determined by qRT-PCR (n = 3, performed in duplicate). *, p < .05; ***, p < .001. Abbreviations: CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; LMPP, lymphoid-primed multipotent progenitor; LT-HSC, long-term hematopoietic stem cell; MEP, megakaryocyte-erythroid progenitor; MPP, multipotent progenitor; MP, myeloid progenitor; ST-HSC, short-term hematopoietic stem cell.

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Alcam-Deficient HSCs Display Robust Myeloid Differentiation and Short-Term Repopulation

Since Alcam is highly expressed in HSCs and subsets of progenitor cells, we used an Alcam-null mouse allele [17] to determine the hematopoietic consequences of Alcam deletion. First, we assessed whether Alcam heterozygosity or Alcam deficiency leads to alteration of the hematopoietic profile in the peripheral blood (PB) of young (2-month old) adult mice. Complete blood count analysis showed no significant changes in hematological parameters for WT, Alcam+/− (HT) or Alcam−/− (KO) mice other than slightly reduced leukocyte counts (p = .0498) in Alcam−/− (KO) (supporting information Table 2). The frequencies of B cells (B220+), T cells (CD3+), and myeloid cells (CD11b+ or Gr1+), as determined by flow cytometry, were not altered in Alcam+/− or Alcam−/− mice (Fig. 2A). The cellularity and frequencies of various HSPC subsets were similar in Alcam+/− or Alcam−/− BM (Fig. 2B). The frequencies of phenotypic progenitor subsets, including LSK (Lin/ckithi/Sca1+), LMPP, CLP, CMP, GMP, and MEP, were similar in Alcam+/− or Alcam−/− mice as compared to WT littermates (Fig. 2C, 2D). There was no significant difference in the frequencies of LT-HSCs, ST-HSCs, and MPPs within the LSK fraction (Fig. 2E). Together, these results indicate that Alcam deficiency does not alter the steady state hematopoietic profile in young adult mice.

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Figure 2. Alcam-deficient HSCs and early progenitors have normal differentiation and proliferation but are compromised in serial-replating capacity. (A): Frequencies of B cells (B220+), T cells (CD3+), and myeloid cells (Gr1+ or CD11b+), in PB, as determined by immunostaining and flow cytometry. (B): Total BM mononuclear cell number isolated from one femur and two tibias of mice. Frequencies of LSKs, LMPPs, CLPs (C); myeloid progenitor subsets (D); and LT-HSCs, ST-HSCs, and MPPs (E) in young (2-month-old) adult BM. For (A–E), bar graphs represent average ± SEM (WT, n = 5; HT, n = 5; KO, n = 6). (F): Bars and numbers indicate percent of Meg/E, GM, G, and Mix colonies identified by single cell differentiation assay (KO, n = 273; WT, n = 267). (G): Percent of Meg/E, GM, and Mix colonies derived from Alcam+ CMPs and Alcam CMPs in CFU-C assay. (H): Colony-forming progenitor (CFU-C) number per 1,000 cells in each sorted population. Bar graphs show representative average ± SEM of duplicate assays from three independent experiments. (I): CFU-C number per 10,000 cells replated over three rounds of weekly successive replating (P2, P3, P4). Bar graphs show representative average ± SEM of duplicate assays from three independent experiments. Bar graphs represent average ± SEM (n = 3). ***, p < .001; ns, not significant. Abbreviations: BM, bone marrow; CFU-C, colony-forming unit; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; GM, granulocyte-macrophage; G, granulocyte; HT, Alcam+/−; KO, Alcam−/−; LT-HSC, long-term hematopoietic stem cell; LSK, Lin/ckithi/Sca1+; LMPP, lymphoid-primed multipotent progenitor; MEP, megakaryocyte-erythroid progenitor; MPP, multipotent progenitor; MP, myeloid progenitor; Meg/E, megakaryocyte-erythroid; Mix, Meg/E and GM; PB, peripheral blood; ST-HSC, short-term hematopoietic stem cell; WT, wild type.

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To assess the consequences of Alcam deficiency on the differentiation potential of HSCs at high resolution, we used a single-cell in vitro differentiation assay [35]. We sorted phenotypic LT-HSCs and plated single cells in erythromyeloid differentiation-inducing media and cytokines (see supporting information Material and Method). We determined the lineage composition by FACS analysis (c-Kit, CD11b, Gr1, Ter119) and morphological examination of cytospin preparations. Overall, Alcam−/− (n = 273) and WT (n = 267) HSCs gave rise to similar phenotypes and numbers of all colony types, including megakaryocyte-erythroid (Meg/E), granulocyte-macrophage (GM), granulocyte (G), and mixed (Mix; contained both Meg/E and GM) (Fig. 2F). Although not statistically significant, we found that Alcam−/− HSCs have enhanced propensity to differentiate into granulocytes (12% in KO compared to 5% in WT), which corresponded to a reduction in immature mixed (Mix) colonies (35% in KO compared to 42% in WT). Since Alcam is expressed in a subset of CMPs, we tested whether they represent functionally distinct progenitors. We performed a colony-forming unit (CFU-C) assay (See supporting information Material and Method) using sorted Alcam+ CMPs and Alcam CMPs (supporting information Fig. 1A, top). We found that while the total CFU-C numbers are similar (data not shown), Alcam+ CMPs are significantly biased toward GM potential (31% for Alcam+ vs. 2% for Alcam) whereas Alcam CMPs are significantly skewed toward Meg/E lineage (38% for Alcam vs. 0% for Alcam+) potential (Fig. 2G; p < .0001). In addition, we assessed Alcam surface expression in refined Pre-GM and Pre-MegE subsets within CMP based on additional markers previously described [36] (supporting information Fig. 1A, bottom). Consistent with the functional bias, Alcam expression was high in Pre-GM and low in Pre-MegE (supporting information Fig. 1A, bottom). These results indicate that Alcam expression in myeloid precursors differentially marks cells with a GM lineage potential. We also assessed clonogenic progenitor activity of sorted LT-HSC, MPP, and MP cells. Alcam−/− LT-HSCs, MPPs, MPs formed similar numbers of colonies as their WT counterparts (Fig. 2H). To assess the ability to maintain self-renewing progenitors, every 7 days we serially replated colonies derived from LT-HSCs and observed a significant reduction in Alcam−/− CFU-Cs upon replating (Fig. 2I; p = .0002). To examine whether Alcam deficiency leads to altered proliferation of stem and progenitor cells, we performed in vivo 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays (see supporting information Materials and Methods). We observed comparable cell cycle distribution for each phenotypic population from Alcam−/− or WT mice (supporting information Fig. 2A–2C), suggesting that Alcam deficiency does not alter the proliferation rate. Expression of cell cycle-related genes such as Cdkn2c (p18), Cdkn1b (p27), p53, and Gata2 were similar in Alcam−/− and WT LT-HSCs (supporting information Fig. 2F–2I).

To further assess the hematopoietic consequences of Alcam deficiency in vivo, we performed a competitive repopulation assay using unfractionated BM cells (Fig. 3A). We compared the relative contribution of CD45.2+ test donor cells to that of congenic competitor cells (CD45.1+) over time. Overall, there was no significant difference in donor chimerism over 16 weeks (Fig. 3B). The contribution of peripheral B cells, T cells, and myeloid cells did not significantly differ between Alcam−/−, Alcam+/−, and WT donors (Fig. 3C); nor did the numbers of donor LT-HSCs (Fig. 3D) and progenitor subsets in the BM (16 weeks) significantly differ (Fig. 3E). We further assessed the long-term self-renewal potential by secondary transplantation. Sorted CD45.2+ LSK cells (2,000 cells) were transplanted together with 2 × 105 CD45.1+ radioprotective cells into CD45.1+ congenic recipients (n = 8), and CD45.2+ donor chimerism was analyzed over 16 weeks. Engraftment was considered positive if CD45.2+ chimerism in all lineages (B, T, myeloid) was greater than 1%. The number of mice showing multilineage engraftment over time was significantly reduced in groups received Alcam−/− (p = .0018) compared to those recieved WT donor cells (Fig. 3F). These results suggest that while Alcam-deficient cells can support short-term repopulation, long-term repopulating activity is compromised.

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Figure 3. Alcam-deficient HSCs display robust short-term repopulation. (A): Schematic representation of competitive repopulation assay design. Unfractionated bone marrow (BM) cells (2 × 105) from 2-month-old Alcam−/− (KO), Alcam+/− (HT), or WT littermates (CD45.2+) were transplanted along with equal numbers of congenic (CD45.1+) BM cells as competitors into recipient mice (CD45.1+/CD45.2+) irradiated at 13 Gy. Donor engraftment in PB was analyzed every 4 weeks, and BM engraftment was assessed at 16 weeks. CD45.2+ LSK cells were then sorted and similarly transplanted along with 2 × 105 congenic (CD45.1+) BM cells into secondary recipients (CD45.1+) irradiated at 13 Gy. (B): Time course analysis of CD45.2+ to CD45.1+ cell ratio in PB of primary competitive repopulation recipients. (C): CD45.2+ to CD45.1+ ratio in peripheral B cells (B220+), T cells (CD3+), and myeloid cells (Gr1+ or CD11b+) of primary recipients at 16 weeks. Each dot represents an individual mouse transplanted with WT (n = 7), HT (n = 8), or KO (n = 8) cells. (D): The number of CD45.2+ LT-HSCs detected in the BM of primary recipients at 16 weeks. Bar graph represents average ± SEM (WT, n = 4; HT, n = 5; KO, n = 5). (E): The number of CD45.2+ ST-HSC, MPP, and LSK cells in the BM at 16 weeks. Bar graph represents average ± SEM (WT, n = 4; HT, n = 5; KO, n = 5). (F): Percentage of mice engrafted with CD45.2+ cells after secondary transplantation (n = 8). Engraftment was considered positive if CD45.2+ chimerism in all lineages (B, T, myeloid) was greater than 1%. **, p < .01; ns, not significant. Abbreviations: HT, Alcam+/−; HSC, hematopoietic stem cell; KO, Alcam−/−; LSK, Lin/ckithi/Sca1+; LT-HSC, long-term hematopoietic stem cell; MPP, multipotent progenitor; PB, peripheral blood; ST-HSC, short-term hematopoietic stem cell; WT, wild type.

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Alcam-Deficient BM Contains Lower Frequency of Long-Term Repopulating Cells

To determine the frequency of functional long-term repopulating cells in Alcam−/− BM, we performed a limiting-dilution transplantation experiment using decreasing numbers (2 × 105, 4 × 104, 8 × 103) of CD45.2+ donor cells together with 2 × 105 CD45.1+ radioprotective cells (supporting information Fig. 3A). Consistent with the competitive repopulation results, all mice transplanted with 2 × 105 cells showed multilineage engraftment up to 16 weeks post-transplant, and there was no significant difference in CD45.2+ chimerism between Alcam−/− and WT donors (supporting information Fig. 3B). Time course analysis showed that overall CD45.2+ chimerism in mice transplanted with lower Alcam−/− donor cell doses was significantly lower (supporting information Fig. 3C, 3D). We analyzed CD45.2+ engraftment, which is considered positive if chimerism is greater than 1% in all lineages (B, T, myeloid) 16 weeks after transplantation. The number of mice showing multilineage engraftment in each cell dose is summarized in Table 1. The results show that engraftment of Alcam−/− cells was significantly (p = .0036) reduced at lower donor cell doses. Based on the fraction of mice engrafted at each cell number, the estimated frequency of functional long-term repopulating cells in Alcam−/− mice was significantly lower (1 in 51,885; range 1/35,999–1/74,781) than in WT mice (1 in 15,213; range 1/10,559–1/21,918). These results indicate that Alcam−/− BM contains approximately threefold lower frequency of long-term repopulating cells.

Table 1. Estimated long-term repopulating cell frequency in Alcam-deficient bone marrow assessed by limiting-dilution transplantation
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Alcam-Deficient LT-HSCs Are Compromised in Long-Term Multilineage Engraftment Potential

Based on our phenotypic analysis and estimate of long-term repopulation cell frequency, we predicted that the long-term repopulation potential and self-renewal of Alcam−/− LT-HSCs could be compromised on a per cell basis. To test this, we transplanted 50 sorted phenotypic LT-HSCs from young Alcam−/− or WT mice (CD45.2+) together with 2 × 105 CD45.1+ support cells into lethally irradiated CD45.1+ congenic recipients (n = 12) (Fig. 4A). Mice that received Alcam−/− LT-HSCs displayed significantly reduced (p < .0001) CD45.2+ chimerism up to 24 weeks post-transplantation (Fig. 4B). Compared to WT transplanted mice, which all showed multilineage CD45.2+ engraftment, only 8 of 12 Alcam−/− transplanted mice were CD45.2+ engrafted at 24 weeks (Fig. 4D, p = .0079). For the mice that had CD45.2+ engraftment at 24 weeks after transplantation, the numbers of Alcam−/− LT-HSCs in the BM tends to be higher than WT but not statistically different (Fig. 4C).

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Figure 4. Alcam−/− LT-HSCs are compromised in long-term multilineage engraftment. (A): Schematic of LT-HSC long-term repopulation assay. LT-HSCs were sorted from WT or KO mice (CD45.2+), and 50 cells were transplanted into each CD45.1+ congenic recipient (n = 12) along with 2 × 105 CD45.1+ radioprotective cells. CD45.2+ chimerism was assessed over 24 weeks, and BM HSC engraftment was analyzed at 24 weeks. CD45.2+ LT-HSCs were then sorted from primary recipients (1°), and 100 LT-HSCs were similarly transplanted into each secondary recipient (2°). Donor chimerism was similarly assessed over time and HSC engraftment in the BM was examined at 24 weeks. (B): Time course of CD45.2+ donor chimerism after primary transplantation. (C): The number of CD45.2+ LT-HSCs, ST-HSCs, MPPs in the BM after primary transplantation (n = 3). (D): Percentage of mice engrafted with CD45.2+ donor cells through 1°- and 2°-transplantation. (E): Time course analysis of CD45.2+ donor chimerism after secondary transplantation (WT: n = 6; KO: n = 5). (F): The number of CD45.2+ LT-HSCs, ST-HSCs, MPPs in the BM after secondary transplantation (WT: n = 6; KO: n = 5). All bar graphs represent mean ± SEM. (G): Representative images of carboxyfluorescein succinimidyl ester (CFSE)-labeled WT (left) or KO (right) LSK cells (white arrow) localized close to the endosteum (indicated by red dash line) in the trabecular bone. Scale bar = 25 μm. *, p < .05; **, p < .01; ns, not significant. Abbreviations: BM HSC, bone marrow hematopoietic stem cell; KO, Alcam−/−; LT-HSC, long-term hematopoietic stem cell; MPP, multipotent progenitor; PB, peripheral blood; ST-HSC, short-term hematopoietic stem cell; WT, wild type.

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To further evaluate the long-term repopulation potential and self-renewal capacity of LT-HSCs, we performed a secondary transplantation of sorted CD45.2+ LT-HSCs (100 cells per mouse) along with 2 × 105 CD45.1+ support cells (Fig. 4A). Time course analysis of secondary recipients showed that Alcam−/− LT-HSC-derived chimerism was significantly less (p < .0001) than that of WT LT-HSCs (Fig. 4E). Likewise, the fraction of multilineage Alcam−/− engrafted mice (one out of five) was markedly lower than that of WT (four out of six) at 24 weeks (Fig. 4D; p = .0079). Although Alcam−/− LT-HSCs had reduced engraftment potential, we found significantly more (p = .0107) phenotypic Alcam−/− LT-HSCs in the BM than WT LT-HSCs (Fig. 4F). Since engraftment of transplanted HSCs require homing to the BM cavity, we tested the homing ability of Alcam−/− HSCs by a short-term homing assay (see supporting information Materials and Methods). The number of labeled Alcam−/− LSK cells in the BM and spleen of transplanted mice 4 hours after transplantation were similar to WT LSK cells (supporting information Fig. 4). We also tested whether Alcam-deficiency alters lodgment of carboxyfluorescein succinimidyl ester (CFSE) labeled LSK cells (40,000 cells per mouse) into nonirradiated recipients 16 hours after injection (see supporting information Materials and Methods). Overall, Alcam−/− LSK cells are capable of localizing close to the endosteum both in the midsections of the long bone and the trabecular bone where HSCs have been shown to preferentially localize [37] (Fig. 4G). In the trabecular bone region, Alcam−/− LSK cells appear to distribute somewhat closer to the endosteum compared to WT LSK (supporting information Fig. 4C; p = .0319). Furthermore, we tested whether Alcam−/− HSCs show enhanced mobilization induced by granulocyte-colony stimulating factor (G-CSF) treatment (300 μg/kg, 5 days). We find that the frequency of phenotypic LT-HSC and ST-HSC in BM, spleen, and PB were similar (supporting information Fig. 5A–5C), and the numbers of CFU-C were not significantly different between Alcam−/− and WT mice (supporting information Fig. 5D). Collectively, these results provide direct evidence that despite the progressive increase in phenotypic LT-HSC population over time, Alcam−/− LT-HSCs are defective in long-term multilineage engraftment which is not caused by inefficient BM homing, lodgment, or enhanced HSC mobilization.

Alcam Deficiency Leads to Age-Associated Increase in LT-HSC Cellularity and Premature Upregulation of Selp

To assess whether Alcam might regulate hematopoiesis in an age-dependent manner, we analyzed the hematopoietic compartment in the BM of 12-month-old Alcam−/− and WT mice. Cellularity in the BM of Alcam−/− mice did not differ significantly from that of WT mice (Fig. 5A), and the frequencies of myeloid and lymphoid progenitors were also comparable (Fig. 5B, 5C). Evaluation of the LSK population revealed that the LT-HSC subset was significantly increased (p = .0288) in 12-month-old Alcam−/− mice (Fig. 5D, 5E). Consistent with the reported age-associated increase in phenotypic HSCs, the frequency of LT-HSCs in BM was twofold greater in 12-month-old WT mice (0.035% ± 0.006%) compared to young (2-month old) WT mice (0.015% ± 0.002%) (Figs. 2E and 5E). In comparison, the LT-HSC frequency in Alcam−/− 12-month-old mice (0.065% ± 0.009%) was fourfold greater than in young Alcam−/− mice (0.016% ± 0.002%). Similar to young LT-HSCs, there was no significant difference in the cell cycle distribution of older Alcam−/− LT-HSCs compared to WT LT-HSCs of the same age shown by EdU incorporation (supporting information Fig. 2D, 2E).

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Figure 5. Alcam deficiency leads to age-associated expansion of LT-HSCs and reduction in leukocyte output. (A): BM cellularity in 12-month-old WT (n = 6) or KO (n = 5) mice. Shown are total numbers of mononuclear cells pooled from one femur and two tibias. Frequencies of CMP, GMP, and MEP (B); and CLP, LMPP, and LSK (C) subsets in the BM. (D): Representative fluorescence-activated cell sorting profiles and gating strategy for phenotypic HSC subsets (LT-HSC, ST-HSC) and CD150hi, CD150lo fractions of LT-HSCs. Numbers shown are the mean frequencies of the cell types in BM of WT (top, n = 6) or KO (bottom, n = 5) mice. (E): Frequencies of LT-HSC, ST-HSC, and MPP subsets in the BM. (F): Frequencies of the CD150hi and CD150lo subsets of LT-HSCs in WT and KO mice. All bar graphs represent mean ± SEM (n = 5 or 6). (G): WBC counts of 2- and 12-month-old WT and KO mice. Differential counts of lymphocytes (LY) (H), neutrophils (NE) (I), monocytes (MO) (J). Each dot represents an individual mouse, lines indicate the median (WT: n = 11; KO: n = 14). *, p < .05; **, p < .01. Abbreviations: BM, bone marrow; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; HSC, hematopoietic stem cell; KO, Alcam−/−; LSK, Lin/ckithi/Sca1+; LMPP, lymphoid-primed multipotent progenitor; LT-HSC, long-term HSC; LY, lymphocytes; MEP, megakaryocyte-erythroid progenitor; MPP, multipotent progenitor; MO, monocytes; Ne, neutrophils; ST-HSC, short-term HSC; WBC, white blood cell; WT, wild type.

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The increase in LT-HSCs seen in older Alcam−/− mice was mostly due to an increase in the CD150hi fraction (Fig. 5D, 5F, KO: 0.048% ± 0.005% compared to WT: 0.024% ± 0.004%; p = .0072), which was reportedly myeloid-biased [10]. Therefore, we assessed whether there was an age-associated alteration of the blood counts in Alcam−/− mice. We found that leukocyte counts were significantly reduced in 12-month-old Alcam−/− mice (Fig. 5G, p = .0012). Differential counts revealed that this decrease was mainly due to a prominent reduction in the number of lymphocytes (Fig. 5H, p = .0020). The numbers of neutrophils (Fig. 5I, p = .0209) and monocytes (Fig. 5J; p = .0186) were also significantly lower in Alcam−/− mice. There was no significant difference in erythrocyte, hematocrit, hemoglobin, or platelet counts (supporting information Fig. 6A–6D) or the lineage composition of leukocytes in the blood or BM in Alcam−/− mice compared to WT by FACS analysis (supporting information Fig. 6E, 6F). We performed a LT-HSC repopulation assay using 100 sorted LT-HSCs from 15-month-old Alcam−/− or WT mice (CD45.2+). Although overall Alcam−/− or WT-derived CD45.2+ chimerism in the PB was similar (Fig. 6A), we observed a significant (p = .0182) biased toward the myeloid lineage within Alcam−/− derived CD45.2+ population in the PB and the BM (Fig. 6B and data not shown). Furthermore, there were significantly (p = .0426) more phenotypic LT-HSCs derived from Alcam−/− in the transplanted BM 24 weeks after transplantation compared to those derived from WT (Fig. 6C). These results are consistent with an age-associated increase of phenotypic LT-HSCs in Alcam−/− mice.

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Figure 6. Alcam deficiency causes cell autonomous increase of long-term hematopoietic stem cell (LT-HSC) with age and premature upregulation of age-related gene expression. (A): Time course analysis of CD45.2+ chimerism in peripheral blood (PB) of mice transplanted with LT-HSCs (100 cells) sorted from 15-month-old WT or KO mice (CD45.2+) along with 2 × 105 CD45.1+ supportive cells. (B): Percentage of B cells (B220+), T cells (CD3+), and myeloid cells (Gr1+ and/or CD11b+) within CD45.2+ donor derived BM cells at 24 weeks after LT-HSC transplantation (n = 7). (C): Number of CD45.2+ LT-HSC in the BM of transplanted mice 24 weeks after LT-HSC transplantation. Each dot represents an individual mouse, lines indicate the median. Relative mRNA levels of Selp(D), Clu(E), Cdc42(F), and Foxo3(G) in sorted LT-HSC from 2-month- and 12- or 15-month-old WT or KO determined by quantitative reverse transcriptase polymerase chain reaction. Bar graph represents mean + SEM (n = 2–4, each in duplicates). *, p < .05. Abbreviations: KO, Alcam−/−; WT, wild type.

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To dissect the molecular mechanism underlying the aging-like phenotype, we assessed changes in expression of genes known to associate with age or are functionally important for HSC aging in sorted LT-HSCs from 2-month-old and 12- or 15-month-old Alcam−/− or WT mice. We analyzed age-related genes including Selp [9, 11, 38], Clu [39], Cdc42 [40], and Foxo3 [41] by qRT-PCR. Consistent with the reported progressive upregulation with age, we detected markedly increased expression of these genes in 12–15 month-old WT LT-HSCs compared to those of 2 months old (Fig. 6D–6G). In Alcam−/− LT-HSCs, expression levels of Selp, Clu, Cdc42, and Foxo3 are substantially elevated at 2 months of age while similar levels are detected at an older age (12 or 15 months) (Fig. 6D–6G). Taken together, these results indicate that Alcam deficiency leads to an age-associated increase in CD150hi phenotypic LT-HSCs and premature upregulation of age-related gene expression.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTERESTS
  10. REFERENCES
  11. Supporting Information

ALCAM is known to be expressed on primitive human HSCs [29, 30], however, its functional contribution to HSC biology has been unclear. This study provides a comprehensive characterization of the role of Alcam in regulating adult HSC function. We report that Alcam is most highly expressed in the primitive subsets of the hematopoietic hierarchy, and its expression is upregulated in LT-HSCs with age. In addition to being highly expressed in HSCs, Alcam is differentially expressed during MP specification. We demonstrate that high Alcam expression in CMPs enriches for progenitors with GM potential whereas low Alcam expression enriches for those with MegE potential (Fig. 2G), consistent with Alcam levels observed in Pre-GM and Pre-MegE subsets (supporting information Fig. 1A). In agreement with these findings, it was recently reported that downregulation of Alcam is necessary for megakaryocytic differentiation [33], underscoring the biological significance of differential gene expression.

Our studies indicate that Alcam deficiency leads to a significantly reduced frequency of long-term repopulating cells, and that Alcam-deficient HSCs are functionally compromised in long-term engraftment on a per cell basis. Analysis of cell proliferation by EdU incorporation showed that cell cycle distribution is not altered in Alcam−/− HSCs or progenitors, regardless of age (supporting information Fig. 2). Thus, Alcam does not appear to regulate general proliferation rate or cell cycle progression of HSCs on a population level. We tested the possibility that Alcam−/− HSCs are defective in homing or lodgment in the BM cavity which may contribute to the engraftment deficiency. We found that Alcam−/− LSK cells had similar homing abilities as WT LSK cells (supporting information Fig. 4A, 4B) and Alcam−/− LSK cells was capable of lodging close to the endosteum (Fig. 4G). We also show that HSC mobilization induced by G-CSF was not affected in Alcam−/− mice (supporting information Fig. 5). Therefore, the engraftment deficiency of Alcam−/− HSCs is not due to inefficient homing, lodgment, or altered mobilization. We have, however, observed significantly higher proportions of Alcam−/− LSK cells located near the endosteum in the trabecular bone region (supporting information Fig. 4C). Alcam is expressed in multiple cell types that constitute the BM microenvironment, including osteoblasts [42], endothelial cells [18], adipose derived stromal cells [43], and perichondrial mesenchymal stem cells [44]. Furthermore, an Alcam+ subset of the endosteal population has recently been shown to support HSC activity through cell adhesion [34]. It is therefore possible that Alcam might regulate the composition of BM microenvironment or the localization of specialized HSC niche components. Whether Alcam deficiency leads to alterations in the HSC niche and the microenvironmental consequences of Alcam loss remain outstanding questions. Further understanding of the microenvironmental composition and functional characteristics of these components is needed to address these questions.

In spite of the reduced long-term engraftment capacity measured in Alcam−/− LT-HSCs, the number of Alcam−/− LT-HSCs was remarkably higher than their WT counterparts after two rounds of long-term repopulation (6 months each) (Fig. 4F). This finding is consistent with increased numbers of phenotypic LT-HSCs detected in 12-month-old Alcam−/− mice (Fig. 5E). Therefore, Alcam deficiency causes cell autonomous, age-associated increase in phenotypic LT-HSC. A series of recent studies have demonstrated that the HSC compartment consists of several subpopulations of HSCs with intrinsically distinct lineage differentiation and proliferation behaviors, and that lymphoid-biased HSCs predominate in early development through young adulthood, whereas myeloid-biased HSCs become progressively dominant during aging [12, 45–48]. The HSC expansion found in 12–15 months old Alcam−/− mice is primarily within the CD150hi subset of HSCs (Fig. 5F), which is associated with the myeloid-biased HSCs predominating the HSC pool with age [10]. Indeed, we found that these Alcam−/− LT-HSCs showed myeloid reconstitution bias when compared to age matched WT LT-HSCs in an in vivo repopulation assay (Fig. 6B). We did not detect apparent changes in relative lineage composition in the PB or BM of older Alcam−/− mice (supporting information Fig. 6E, 6F). This is perhaps not surprising, since it has been shown that despite a clear shift in HSC subtype composition with age in the C57Bl/6 strain of mice, there is no significant change in the blood cell lineage ratio [47]. It has been suggested that a threshold may exist under which homeostatic mechanisms can overcome the differentiation bias of HSCs.

The mechanism underlying age-associated phenotypic HSC expansion is not well understood. Based on our study and the report that myeloid-biased HSCs are able to self-renew more extensively than lymphoid-biased HSCs [48], it is tempting to speculate that the HSC composition in Alcam−/− mice might shift progressively more toward myeloid-biased with age. We show that Alcam levels on LT-HSC cell surface increases with age (Fig. 1C). Conceivably, Alcam might be upregulated to provide a protective mechanism counteracting HSC aging and maintain a balanced HSC composition. Gene ontology analysis of age-associated gene expression found that Nuclear Factor-kappa B (NF-κB) signaling was the most significantly enriched category activated in HSCs during aging [11]. Meanwhile, NF-κB reportedly regulates ALCAM transcription through direct binding to the NF-κB response elements in the proximal promoter [49]. Whether similar regulatory mechanism by NF-κB is conserved in the mouse remains to be determined. We speculate that age-related upregulation of NF-κB signaling might underlie the increased expression of Alcam with age (Fig. 1E). In addition, we find that Alcam deficiency leads to a substantial increase of age-associated gene [9, 11, 38, 50] including Selp, Clu, Cdc42, and Foxo3 in LT-HSCs at a young age (Fig. 6D–6G). Upregulation of Selp, which encodes P-selectin, is among the most significant age-associated alteration commonly found during HSC aging [9, 11, 38]. Loss of P-selectin has been shown to result in increased HSC self-renewal and enhanced chronic myeloid leukemia transformation, suggesting that P-selectin might negatively regulate HSC self-renewal [38]. Foxo3, a forkhead transcription factor, is essential for maintenance of HSC quiescence and HSC pool with age [41]. Based on our finding, regulation of HSC pool might be disrupted in Alcam−/− mice in part through increased expression of Foxo3 and premature upregulation of P-selectin. Recently, elevated activity of the small RhoGTPase Cdc42 in aged HSCs is linked to a loss of cell polarity and HSC aging [40]. Consistent with an elevated Cdc42 activity in aged HSCs, we found that Cdc42 expression in HSCs increases with age and that Cdc42 level is prematurely elevated in Alcam−/− HSCs. These results suggest that Alcam deficiency might lead to early loss of HSC cell polarity through activation of Cdc42. It has long been suggested that altered interactions with the BM stroma might cause functional changes in aging HSCs [51, 52]. This study provides direct evidence that Alcam-mediated cell adhesion is cell autonomously required for maintaining a functional HSC pool and a controlled HSC pool size during transplantation and aging.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTERESTS
  10. REFERENCES
  11. Supporting Information

Alcam is a cell adhesion molecule that is highly expressed in adult LT-HSCs where its expression progressively increases with age. This study provides evidence that Alcam regulates the functional integrity and self-renewal of LT-HSCs upon transplantation and during aging. This work reveals previously unappreciated functions of Alcam in HSC biology, with important implications in hematopoietic transplantation. Further investigation of Alcam signaling effectors, and its role in cell polarity and malignant transformation will be important for the evaluation of potential translational opportunities.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTERESTS
  10. REFERENCES
  11. Supporting Information

This work was supported by research grants from the Margaret E. Early Medical Research Trust (to Y.-H.K.), STOP CANCER Foundation (to Y.-H.K.), Concern Foundation CONquer canCER now award (to Y.-H.K.), the V Foundation V scholar award (to Y.-H.K.), the American Cancer Society Research Scholar Grant 123278-RSG-12-140-01-CSM (to Y.-H. K), and by the National Cancer Institute Grant P30 CA033572 (to City of Hope). R.J. is supported by a California Institute of Regenerative Medicine training grant TG2-01150 (to City of Hope). We thank Joshua Weiner (University of Iowa) for the generous gift of the Alcam-null allele, and the excellent technical support of the Analytical Cytometry Core and Animal Resource Center at City of Hope. We thank Keely Walker for critical review and editorial assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTERESTS
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTERESTS
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
sc-12-0678_sm_SupplData.pdf59KSupplementary Data
sc-12-0678_sm_SupplFigure1a.pdf553KSupplementary Figure 1. Alcam expression marks a subset of CMP cells. (A) Representative FACS plot of sorting Alcam+ CMP and Alcam- CMP subsets (top) and enrichment of PreMegE and PreGM subsets using additional markers including CD105 and CD150 (bottom). (B) Representative FACS plot (top) and morphology of cytospin preparation (bottom) for each type of colonies (Mixed, MegE and GM).
sc-12-0678_sm_SupplFigure1b.pdf429KSupplementary Figure 1. Alcam expression marks a subset of CMP cells. (A) Representative FACS plot of sorting Alcam+ CMP and Alcam- CMP subsets (top) and enrichment of PreMegE and PreGM subsets using additional markers including CD105 and CD150 (bottom). (B) Representative FACS plot (top) and morphology of cytospin preparation (bottom) for each type of colonies (Mixed, MegE and GM).
sc-12-0678_sm_SupplFigure2a.tif2024KSupplementary Figure 2. Alcam deficiency does not alter cell cycle distribution of hematopoietic stem and progenitor cells. (A) Representative FACS plot of EdU and DAPI staining. Shown in each gate are representative frequencies of indicated populations (LT- HSCs, MPPs or MPs) in S and G0- G1, G2- M phase of the cell cycle. Frequencies in S (B) and G0- G1 (C) phase of the cell cycle for LT- HSCs, MPPs or MPs from 2- month old mice. Frequencies in S (D) and G0- G1 (E) phase of the cell cycle for LT- HSCs, MPPs or MPs from12- month old mice. Bar graphs represent average +/− SEM (n=3). Relative mRNA expression levels of Cdkn2c (F), Cdkn1b (G), p53 (H) and Gata2 (I) in LT- HSC from 2 months old or 12 months old mice, as determined by qRTPCR (n=2- 4).
sc-12-0678_sm_SupplFigure2b.tif503KSupplementary Figure 2. Alcam deficiency does not alter cell cycle distribution of hematopoietic stem and progenitor cells. (A) Representative FACS plot of EdU and DAPI staining. Shown in each gate are representative frequencies of indicated populations (LT- HSCs, MPPs or MPs) in S and G0- G1, G2- M phase of the cell cycle. Frequencies in S (B) and G0- G1 (C) phase of the cell cycle for LT- HSCs, MPPs or MPs from 2- month old mice. Frequencies in S (D) and G0- G1 (E) phase of the cell cycle for LT- HSCs, MPPs or MPs from12- month old mice. Bar graphs represent average +/− SEM (n=3). Relative mRNA expression levels of Cdkn2c (F), Cdkn1b (G), p53 (H) and Gata2 (I) in LT- HSC from 2 months old or 12 months old mice, as determined by qRTPCR (n=2- 4).
sc-12-0678_sm_SupplFigure2c.tif92KSupplementary Figure 2. Alcam deficiency does not alter cell cycle distribution of hematopoietic stem and progenitor cells. (A) Representative FACS plot of EdU and DAPI staining. Shown in each gate are representative frequencies of indicated populations (LT- HSCs, MPPs or MPs) in S and G0- G1, G2- M phase of the cell cycle. Frequencies in S (B) and G0- G1 (C) phase of the cell cycle for LT- HSCs, MPPs or MPs from 2- month old mice. Frequencies in S (D) and G0- G1 (E) phase of the cell cycle for LT- HSCs, MPPs or MPs from12- month old mice. Bar graphs represent average +/− SEM (n=3). Relative mRNA expression levels of Cdkn2c (F), Cdkn1b (G), p53 (H) and Gata2 (I) in LT- HSC from 2 months old or 12 months old mice, as determined by qRTPCR (n=2- 4).
sc-12-0678_sm_SupplFigure3a.tif658KSupplementary Figure 3. Alcam- deficient cells show robust transient repopulation but reduced long- term engraftment activity. (A) Schematic of limiting- dilution transplantation experimental design. Diluting numbers (2 × 105, 4 × 104, 8 × 103) of unfractionated WT or KO cells (CD45.2+) were transplanted into lethally irradiated CD45.1+ congenic recipients (n=7 or 8) together with JEANNET et al. Alcam controls LT- HSC engraftment & self- renewal 2 × 105 CD45.1+ radio- protective cells. Donor chimerism was assessed every 4 weeks and BM engraftment was analyzed 16 weeks after transplantation. Engraftment was considered positive if CD45.2+ donor- derived B cells (B220+ ), T cells (CD3+ ), and myeloid cells (Gr1+ and/ or CD11b+) in peripheral blood all exceeded 1% at 16 weeks post- transplantation. (B) Time course of CD45.2+ chimerism in mice transplanted with 2 × 105 donor cells (WT: n=7; KO: n=7). (C) Time course of CD45.2+ donor chimerism in mice that received 4 × 104 donor cells (WT: n=8; KO: n=8; p=0.0119). (D) Time course of CD45.2+ donor chimerism in mice that received 8 × 103 donor cells (WT: n=8; KO: n=8).
sc-12-0678_sm_SupplFigure3b.tif350KSupplementary Figure 3. Alcam- deficient cells show robust transient repopulation but reduced long- term engraftment activity. (A) Schematic of limiting- dilution transplantation experimental design. Diluting numbers (2 × 105, 4 × 104, 8 × 103) of unfractionated WT or KO cells (CD45.2+) were transplanted into lethally irradiated CD45.1+ congenic recipients (n=7 or 8) together with JEANNET et al. Alcam controls LT- HSC engraftment & self- renewal 2 × 105 CD45.1+ radio- protective cells. Donor chimerism was assessed every 4 weeks and BM engraftment was analyzed 16 weeks after transplantation. Engraftment was considered positive if CD45.2+ donor- derived B cells (B220+ ), T cells (CD3+ ), and myeloid cells (Gr1+ and/ or CD11b+) in peripheral blood all exceeded 1% at 16 weeks post- transplantation. (B) Time course of CD45.2+ chimerism in mice transplanted with 2 × 105 donor cells (WT: n=7; KO: n=7). (C) Time course of CD45.2+ donor chimerism in mice that received 4 × 104 donor cells (WT: n=8; KO: n=8; p=0.0119). (D) Time course of CD45.2+ donor chimerism in mice that received 8 × 103 donor cells (WT: n=8; KO: n=8).
sc-12-0678_sm_SupplFigure4a.tif1468KSupplementary Figure 4. Alcam- deficient HSCs are able to home to BM and localize to endosteum. (A) Representative FACS plot for gating eFour670- labeled test cells and Violet- labeled input control cells. (B) Relative amount of eFour670- labeled Alcam−/− (KO) or WT LSK cells detected in BM, spleen (SP), peripheral blood (PB) and liver (LV) 4 hours after transplantation. Data was normalized with co- injected input control cells stained with CellTrace Violet (Invitrogen). Each dot represent an individual mouse, lines indicate the median (WT, n=7; KO, n=7). (C) The distance of CFSElabeled LSK cells from the endosteum in the midsection or the trabecular regions of the femur in a lodgment assay. Each dot represents an individual cell (midsection WT: n=20; midsection KO: n=10; trabecular WT: n=22; trabecular KO: n=12). *p < 0.05
sc-12-0678_sm_SupplFigure4b.tif391KSupplementary Figure 4. Alcam- deficient HSCs are able to home to BM and localize to endosteum. (A) Representative FACS plot for gating eFour670- labeled test cells and Violet- labeled input control cells. (B) Relative amount of eFour670- labeled Alcam−/− (KO) or WT LSK cells detected in BM, spleen (SP), peripheral blood (PB) and liver (LV) 4 hours after transplantation. Data was normalized with co- injected input control cells stained with CellTrace Violet (Invitrogen). Each dot represent an individual mouse, lines indicate the median (WT, n=7; KO, n=7). (C) The distance of CFSElabeled LSK cells from the endosteum in the midsection or the trabecular regions of the femur in a lodgment assay. Each dot represents an individual cell (midsection WT: n=20; midsection KO: n=10; trabecular WT: n=22; trabecular KO: n=12). *p < 0.05
sc-12-0678_sm_SupplFigure5a.tif38KSupplementary Figure 5. Alcam- deficiency does not alter G- CSF induced HSC mobilization. Alcam−/− or WT mice were subcutaneously injected with G- CSF JEANNET et al. Alcam controls LT- HSC engraftment & self- renewal (300ug/ kg) for 5 consecutive days and phenotypic HSC subsets were analyzed by flow cytometry 6 hours after the last injection. Frequencies of phenotypic LT- HSC and STHSC in the bone marrow (A), spleen (SP) (B) and peripheral blood(PB) (C) determined by FACS. (D) The number of CFU- C in 1,000 mobilized PB cells. Each dot represent an individual mice, lines indicate the median (n=5).
sc-12-0678_sm_SupplFigure5b.tif34KSupplementary Figure 5. Alcam- deficiency does not alter G- CSF induced HSC mobilization. Alcam−/− or WT mice were subcutaneously injected with G- CSF JEANNET et al. Alcam controls LT- HSC engraftment & self- renewal (300ug/ kg) for 5 consecutive days and phenotypic HSC subsets were analyzed by flow cytometry 6 hours after the last injection. Frequencies of phenotypic LT- HSC and STHSC in the bone marrow (A), spleen (SP) (B) and peripheral blood(PB) (C) determined by FACS. (D) The number of CFU- C in 1,000 mobilized PB cells. Each dot represent an individual mice, lines indicate the median (n=5).
sc-12-0678_sm_SupplFigure6a.tif47KSupplementary Figure 6. Hematopoietic profile of 12- month old mice. Red blood cell (RBC) (A), hematocrit (HCT)(B), Hemoglobin (Hb)(C), and platelets (PLT) counts (D). (E) Frequencies of peripheral B- T- and myeloid cells in the blood determined by FACS. Each dot represents an individual mice, lines indicate the median (WT: n=11; KO: n=14). (F) Frequencies of B- T- and myeloid cells in the bone marrow determined by FACS. (WT: n=5; KO: n=4)
sc-12-0678_sm_SupplFigure6b.tif31KSupplementary Figure 6. Hematopoietic profile of 12- month old mice. Red blood cell (RBC) (A), hematocrit (HCT)(B), Hemoglobin (Hb)(C), and platelets (PLT) counts (D). (E) Frequencies of peripheral B- T- and myeloid cells in the blood determined by FACS. Each dot represents an individual mice, lines indicate the median (WT: n=11; KO: n=14). (F) Frequencies of B- T- and myeloid cells in the bone marrow determined by FACS. (WT: n=5; KO: n=4)
sc-12-0678_sm_SupplTable1.tif752KSupplementary Table 1
sc-12-0678_sm_SupplTable2.tif60KSupplementary Table 1

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