Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Aug 9;6(15):4373-4391.
doi: 10.1182/bloodadvances.2022007465.

Refining the migration and engraftment of short-term and long-term HSCs by enhancing homing-specific adhesion mechanisms

Asma S Al-Amoodi  1 , Yanyan Li  1 , Arwa Al-Ghuneim  1 , Hanaa Allehaibi  1 , Ioannis Isaioglou  1 , Luke E Esau  2 , Dina B AbuSamra  1 , Jasmeen S Merzaban  1
Affiliations

Refining the migration and engraftment of short-term and long-term HSCs by enhancing homing-specific adhesion mechanisms

Asma S Al-Amoodi et al. Blood Adv. .

Abstract

In contrast to the short-term (ST) CD34+ stem cells, studies have suggested that long-term (LT) hematopoietic stem cells (HSCs) found in the CD34- stem cell pool have trouble migrating and engrafting when introduced through IV. To understand why these deficiencies exist, we set out to fully elucidate the adhesion mechanisms used by ST and LT-HSCs to migrate to the bone marrow(BM). Specifically focusing on murine ST-HSCs (Flk2-CD34+) and LT-HSCs (Flk2-CD34-), we observed a distinctive expression pattern of BM homing effectors necessary for the first step, namely sialyl Lewis-X (sLex) (ligand for E-selectin), and the second step, namely CXCR4 chemokine receptor (receptor for SDF-1). sLex expression was higher on Flk2-CD34+ ST-HSCs (>60%) compared with Flk2-CD34- LT-HSCs (<10%), which correlated to binding to E-selectin. Higher concentrations of CXCR4 were observed on Flk2-CD34+ ST-HSCs compared with Flk2-CD34- LT-HSCs. Interestingly, the expression of CD26, a peptidase known to deactivate chemokines (ie, SDF-1), was higher on Flk2-CD34- LT-HSCs. Given that both E-selectin-binding and CXCR4-mediated migration are compromised in Flk2-CD34- LT-HSCs, we aimed to enhance their ability to migrate using recombinant human fucosyltransferase 6 (rhFTVI) and the CD26 inhibitor, Dip A (diprotin A). To this end, we observed that although LT-HSCs expressed low concentrations of sLex, they were able to engraft when transplanted into recipient mice. Moreover, although both CD26 inhibition and fucosylation enhanced migration of both HSC populations in vitro, only pretreatment of LT-HSCs with Dip A enhanced engraftment in vivo after transplantation into recipient mice. Remarkably, fucosylation of Flk2-CD34+ ST-HSCs consistently led to their ability to transplant secondary recipients. These data suggest that using fucosylation and Dip A to overcome the molecular disparity in adhesion mechanisms among ST-HSCs and LT-HSCs differentially influences their abilities to migrate and engraft in vivo and promotes the ability of ST-HSCs to engraft secondary recipient mice, the gold standard for testing functionality of LT-HSCs.

PubMed Disclaimer

Figures

None
Graphical abstract
Figure 1
Figure 1
Sorting and characterization of BM-derived murine Flk2−CD34−LT-HSCs and Flk2−CD34+ ST-HSCs. (A) Expression analysis of (left) stem cell markers in the Lin− fraction of the BM of C57BL/6 mice. Following mononuclear cell isolation from the BM and lineage depletion, the cells were analyzed for the stem cell markers, Sca-1, c-Kit, CD34, and Flk2; and (right) of Flk2 and CD34 following gating on LSK using flow cytometric analysis. Each data point represents mean ± SD. (Bi) Lin− BM cells were stained for c-Kit and Sca-1 along with CD34 and Flk2. (Bii) Following the selection of live-singlet cells, c-Kit and Sca-1 double-positive cells were investigated for their expression of CD34 and Flk2 to isolate Flk2−CD34+ HSCs (purple), Flk2−CD34− HSCs (green), and progenitors (orange). Gates denote the sorting strategy used for each of the populations (n = 29 independent experiments). (C) The frequency of each sorted population of Flk2−CD34− LT-HSCs and Flk2− CD34+ ST-HSCs in the whole BM cells (left) and in the Lin− fraction following lineage depletion (right). (D) RNA was isolated from sorted Flk2−CD34− LT-HCs and Flk2−CD34+ ST-HSCs. SYBR green-based real-time qPCR was carried out using primers for CD34 and glyceraldehyde-3-phosphate (GAPDH). Results from qPCR were obtained from n = 3 independent experiments. (Ei) Both HSC populations were stained with CD150 antibody and gated according to fluorescence minus 1 (FMO) to determine CD150 expression using flow cytometric analysis. (Eii) The graph is representative of 1 experiment from n = 3 independent experiments. Results were quantified and analyzed using the unpaired t test (**P = .001) and presented as mean ± SEM.
Figure 2
Figure 2
Analysis of homing-related molecules on murine Flk2−CD34− LT-HSCs and Flk2−CD34+ ST-HSCs illustrates a difference in the expression of E-selectin ligands and CXCR4. (A) Flow cytometric analysis was used to determine sLex expression on HSC populations using HECA-452-mAb (gray histogram). Isotype controls (rat IgM) are illustrated in the black histograms. E-selectin binding was also assessed by incubating the cells with E-selectin-hIg chimeric protein followed by anti-human IgG-PE (gray histogram). As a control, 10 mM EDTA was added to abrogate the binding of cells to E-selectin (black histogram). (Ai) Representative histograms are shown from n = 5 independent experiments. A statistical analysis was performed with the unpaired t test (**P = .001 and ***P < .001) as illustrated in (Aii). (B) The expression of the integrins, CD49e, CD49d, and CD29, and the chemokine receptor, CXCR4, on HSC populations was analyzed by flow cytometry. Positive expression was determined as the percent positive cells ± SEM compared with FMO control. Statistical analysis was performed using the unpaired t test (**P = .001). (C) Flow cytometric analysis of E-selectin ligands (CD43, CD44, and PSGL-1) expressed on the 2 subsets of HSCs. Results are expressed as an average percent of expression (above the isotype control) of n = 3 independent experiments. (D) Mass spectrometry data analysis of E-selectin ligands were immunoprecipitated using recombinant E-selectin protein from lysates of Flk2−CD34+ and Flk2−CD34− HSCs, and the purified proteins were identified by mass spectrometry. The identified proteins were analyzed using cell adhesion as a biological process in gene ontology and the data plotted according to the P value for each identified protein.
Figure 3
Figure 3
Glycosyltransferases (GTs) important for sLex decorations are differentially expressed between murine Flk2−CD34− LT-HSCs and Flk2−CD34+ ST-HSCs. GT gene expression was assessed using real-time semiquantitative PCR. qPCR was performed as detailed in the Materials and methods section. (A) The level of expression of the murine GT genes, fucosyltransferases (FT), and sialyltransferases (ST) were determined relative to the GADPH housekeeping gene. Each data point represents the mean ± SEM (n = 3 independent experiments) and statistical analysis was performed using the unpaired t test (*P = .033, **P = .002, and ***P < .001). (B) Heatmap generated from qPCR of GT gene expression in the murine HSC populations. Gene expression of the 14 GT genes was determined and hierarchically clustered in a genewise manner according to the correlation distance and average clustering and with the tightest cluster first. ClustViA, a web tool, was used for visualizing the clustering of multivariate data (BETA). The color intensity indicates expression levels where red is the high-expressed and blue is the low-expressed, while gray indicates that it wasn't detected. Each sample was performed in biological triplicates (n = 3).
Figure 4
Figure 4
rhFTVI treatment induces sLex expression on all Lin− stem populations. (A) Cells sorted for Flk2− CD34− LT-HSCs and Flk2− CD34+ ST-HSCs were treated either with rhFTVI or in buffer alone, as outlined in the Materials and methods section. After treatment, flow cytometric analysis for sLex expression (HECA-452) was performed. Plots are representative of n = 4 independent experiments. (B) The average percentage expression for each condition in (A) is represented. (C) The binding of E-selectin-hIg chimera was measured on sorted Flk2−CD34− LT-HSCs and Flk2−CD34+ ST-HSC populations treated either with rhFTVI or in buffer alone using flow cytometric analysis. (D) Colony-forming capacities of sorted Flk2−CD34− LT-HSCs and Flk2−CD34+ ST-HSCs were determined. Five hundred cells were either treated with rhFTVI or untreated and were cultured in methylcellulose in the presence of cytokines (SCF, IL-3, EPO, and GM-CSF) for 12 to 14 days. The number and composition of the colonies generated were counted and represented in the graph. BFU-E, erythroid burst-forming unit; CFU-GEMM, granulocyte-erythroid-megakaryocyte-macrophages colony-forming unit; CFU-GM, granulocyte-macrophage colony-forming unit.
Figure 5
Figure 5
Inhibiting CD26 activity improves the migration of Flk2−CD34− LT-HSCs and Flk2−CD34+ ST-HSCs in vitro. (A) Representative flow cytometric contour plots (n = 3 independent experiments) illustrating CD26 expression on Flk2−CD34− LT-HSCs and Flk2−CD34+ ST-HSCs isolated from the BM of C57BL/6. (B) Data from (A) is presented as percent (%) positive cells ± SEM as compared with FMO control. The statistical analysis was performed with the unpaired t test (**P = .001). (C) RT-PCR was used to determine the expression of CD26 in the Flk2−CD34− LT-HSCs and Flk2−CD34+ ST-HSCs. Results were obtained from n = 3 independent experiments and analyzed using the unpaired t test (*P = .03) and presented as mean ± SEM. (D) The activity of CD26 peptidase in both HSC populations before and after CD26 inhibition with Dip A was assessed from n = 3 independent experiments (*P = .03 and ***P < .001). (E) A transwell migration assay was used to assess the migration of both HSC populations toward SDF-1α with (+) and without (−) the presence of Dip A. Data were normalized by subtracting the percent migration of untreated cells without SDF-1α. Each sample was performed in triplicate, and n = 3 independent experiments were performed.
Figure 6
Figure 6
CD26 inhibition enhanced engraftment of Flk2−CD34− HSCs. (A) Flk2−CD34− LT-HSCs and Flk2−CD34+ ST-HSCs were isolated from the BM of donor mice (CD45.2), either treated with 5mM Dip A, a CD26 inhibitor, or left untreated and IV-injected into lethally irradiated recipient mice (CD45.1). Ten, 18, and 30 days after cell transplantation, BM aspirates from recipient mice were analyzed by flow cytometry to measure the percentage of donor cells (expressing CD45.2) found in recipient tissues. (B) Results of the engraftment of donor cells treated with Dip A [red] and control [blue] in the BM of recipient mice were determined at each time point for both the ST-HSCs (left panels) and the LT-HSCs (right panels). Data presented are the mean ± SD of >5 mice from each group at the different time points (*P = .02). (C) A representative analysis of donor cell engraftment for both ST-HSCs (left panel) and LT-HSCs (right panel) that were either treated with Dip A (red) or left untreated (blue).
Figure 7
Figure 7
Fucosylation of Flk2−CD34− LT-HSCs and Flk2−CD34+ ST-HSCs improves engraftment in primary and secondary recipients differentially. Lethally irradiated Ly5.1 mice were transplanted with ∼500 to 1000 rhFTVI-treated (red) or buffer-treated (blue) Flk2−CD34+ (left panels in (A) and (B) or Flk2−CD34− (right panels in [A] and [B]) HSCs from Ly5.2 donor BM. BM from transplanted recipient mice was then investigated at the indicated (A) short-term periods and (B) longer-term periods for the percentage of donor cell contribution to total BM cells. Each data point represents the mean ± SEM (*P = .033, **P = .002, and ***P < .001). (C) A representative analysis of donor cell engraftment for both Flk2−CD34+ HSCs (left panel) and Flk2−CD34− HSCs (right panel), either buffer-treated (blue) or rhFTVI-treated (red), is shown. (D) Assessment of the contribution of donor cells to the blood lineage cells in the recipient mice following transplantation of buffer-treated and FTVI-treated Flk2−CD34+ and Flk2−CD34− in primary recipient mice. BM was first gated using flow cytometry for the donor cells (CD45.2), and further analysis for blood lineage phenotypes was assessed. For myeloid cells, granulocytes (Gr-1+ and CD11b+) and monocytes (Gr-1− and CD11b+) were assessed, and for lymphoid cells, B-cell (CD19) and T-cell (CD3) subsets were assessed. CD34 was used as a stem cell marker. All data presented are means ± SD of >5 mice from each group. (E) Flk2−CD34+ HSCs were collected from primary transplanted mice and transplanted into irradiated secondary recipients. At the indicated times following transplantation, BM was assessed for the percent of the donor cells in the recipient mouse, and statistical analysis was performed using the unpaired t test (*P = .033).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Donnelly DS, Zelterman D, Sharkis S, Krause DS. Functional activity of murine CD34+ and CD34- hematopoietic stem cell populations. Exp Hematol. 1999;27(5):788–796. - PubMed
    1. Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science. 1996;273(5272):242–245. - PubMed
    1. Goodell MA, Rosenzweig M, Kim H, et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med. 1997;3(12):1337–1345. - PubMed
    1. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med. 1996;183(4):1797–1806. - PMC - PubMed
    1. Adolfsson J, Borge OJ, Bryder D, et al. Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity. 2001;15(4):659–669. - PubMed

Publication types

  NODES
INTERN 1
Note 1
twitter 2