Direct Interaction Between CD34+ Hematopoietic Stem Cells and Mesenchymal Stem Cells Reciprocally Preserves Stemness

Abstract

Background/objectives: A specialized microenvironment in the bone marrow, composed of stromal cells including mesenchymal stem cells (MSCs), supports hematopoietic stem cell (HSC) self-renewal, and differentiation bands play an important role in leukemia development and progression. The reciprocal direct interaction between MSCs and CD34⁺ HSCs under physiological and pathological conditions is yet to be fully characterized.

Methods: Here, we established a direct co-culture model between MSCs and CD34⁺ HSCs or MSCs and acute myeloid leukemia cells (THP-1, Molm-13, and primary cells from patients) to study heterocellular communication.

Results: Following MSCs-CD34⁺ HSCs co-culture, the expression of adhesion markers N-Cadherin and connexin 43 increased in both cell types, forming gap junction channels. Moreover, the clonogenic potential of CD34⁺ HSCs was increased. However, direct contact of acute myeloid leukemia cells with MSCs reduced the expression levels of connexin 43 and N-Cadherin in MSCs. The impairment in gap junction formation may potentially be due to a defect in the acute myeloid leukemia-derived MSCs. Interestingly, CD34⁺ HSCs and acute myeloid leukemia cell lines attenuated MSC osteoblastic differentiation upon prolonged direct cell-cell contact.

Conclusions: In conclusion, under physiological conditions, connexin 43 and N-Cadherin interaction preserves stemness of both CD34⁺ HSCs and MSCs, a process that is compromised in acute myeloid leukemia, pointing to the possible role of gap junctions in modulating stemness.

Keywords: CD34+ hematopoietic stem cells; N-Cadherin; acute myeloid leukemia; bone marrow; connexin 43; gap junction; heterocellular interaction; mesenchymal stem cells; microenvironment.

Figure 1

Direct co-culture of MSCs and CD34⁺ HSCs: (A) Schematic representation of the direct co-culture system between MSCs and CD34⁺ HSCs. Representative images of HSCs alone, MSCs alone, and MSCs + CD34⁺ HSCs after direct co-culture. Scale bar, 100 µm. (B) Histogram representing mRNA expression levels of adhesion and communication markers in MSCs following direct co-culture, assessed by qPCR. * p ≤ 0.05 and ** p ≤ 0.01 (t-test). (C) Western blot of Cx-43 and N-Cad expression in MSCs after 24 h of direct co-culture with CD34⁺ HSCs. Results are represented as normalized expression to GAPDH in three independent experiments ± SEM. ** p ≤ 0.01 (t-test). (D) Expression of adhesion/communication markers and VEGF, CXCR4 in CD34⁺ HSCs following direct co-culture, assessed by qPCR. Results are represented as normalized expression of GAPDH in three independent experiments. * p ≤ 0.05 and ** p ≤ 0.01 (t-test). (E) Clonogenic potential of CD34⁺ HSCs, expressed as the number of colonies after direct co-culture with MSCs. (F) Cell profiling of CD34⁺ HSCs after co-culture, showing the percentage of CD38⁺ and CD45⁺ cells in both suspension and adherent fractions. Green population refers to CD45⁺ cells, red population refers to CD38⁺ cells and purple population refers to CD45⁻CD38⁻ cells.

Figure 2

(A) Double immunostaining of Cx-43 (red), and N-Cad (green) in MSC alone and MSC + CD34⁺ HSC following direct co-culture. Arrows point to the co-localization of the two proteins. Scale bar, 5 µm. (B) Cx-43 and N-Cad interaction in MSCs and HSCs after direct co-culture as detected by Duo-Link assay. The dots (red) are representatives of the close proximity of the two proteins of interest. Nuclei were stained with Hoechst 33,342 dye (blue). Scale bar, 10 µm (upper panel) and 2 µm (lower panel). (C) Representative flow cytometry graph showing the shift in MFI following co-culture of unlabeled HSCs with Calcein-labeled MSCs.

Figure 3

Engagement in osteoblastic differentiation of MSCs after co-culture with HSCs for 24 h or 21 days: (A) Left panel: Expression of ALP protein in MSCs detected by immunofluorescence. Right panel: Alizarin red staining of MSCs visualized by light microscopy. Scale bar, 20 µm. (B) mRNA expression of ALP and Oct-4 in MSC following direct interaction with CD34⁺ HSC for 24 h and 21 d. **** p ≤ 0.0001 (t-test).

Figure 4

Direct co-culture of MSCs and AML cells: (A) Schematic representation of the direct co-culture system between MSCs and THP-1 and Molm-13. Scale bar, 100 µm. (B) mRNA and protein expression levels of Cx-43 and N-Cad in MSCs following direct co-culture with AML cell lines, assessed by qPCR and Western blot, respectively. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 (t-test). (C) Representative flow cytometry graphs showing the shift in MFI following co-culture of unlabeled AML cell lines (THP-1 and Molm-13) with Calcein-labeled MSCs. (D) Fluorescent and light microscopy images of MSCs co-cultured with AML cell lines for ALP expression and Alizarin Red staining, respectively. Scale bar, 20 µm. (E) Expression of Cx-43 and N-Cad in MSCs following direct co-culture with AML primary cells (3 patients), assessed by Western blot analysis. * p ≤ 0.05 (t-test). (F) mRNA expression levels of Cx-43 and N-Cad in MSCs of healthy and AML patients following direct co-culture with AML cell lines, CD34⁺ N from healthy individuals (n = 1) and CD34⁺ AML from patients (n = 2).