Murine very small embryonic-like (VSEL) cells, defined by the Lin − Sca-1 + CD45 − phenotype and small size, were described as pluripotent cells and proposed to be the most primitive hematopoietic precursors in adult bone marrow. Although their isolation and potential application rely entirely on flow cytometry, the immunophenotype of VSELs has not been extensively characterized. Our aim was to analyze the possible heterogeneity of Lin − Sca + CD45 − population and investigate the extent to which VSELs characteristics may overlap with that of hematopoietic stem cells (HSCs) or endothelial progenitor cells (EPCs). The study evidenced that murine Lin − Sca-1 + CD45 − population was heterogeneous in terms of c-Kit and KDR expression. Accordingly, the c-Kit + KDR − , c-Kit − KDR + , and c-Kit − KDR − subpopulations could be distinguished, while c-Kit + KDR + events were very rare. The c-Kit + KDR − subset contained almost solely small cells, meeting the size criterion of VSELs, in contrast to relatively bigger c-Kit − KDR + cells. The c-Kit − KDR − FSC low subset was highly enriched in Annexin V-positive, apoptotic cells, hence omitted from further analysis. Importantly, using qRT-PCR, we evidenced lack of Oct-4A and Oct-4B mRNA expression either in whole adult murine bone marrow or in the sorted of Lin − Sca-1 + CD45 − FSC low population, even by single-cell qRT-PCR. We also found that the Lin − Sca-1 + CD45 − c-Kit + subset did not exhibit hematopoietic potential in a single cell-derived colony in vitro assay, although it comprised the Sca-1 + c-Kit + Lin − (SKL) CD34 − CD45 − CD105 + cells, expressing particular HSC markers. Co-culture of Lin − Sca-1 + CD45 − FSC low with OP9 cells did not induce hematopoietic potential. Further investigation revealed that SKL CD45 − CD105 + subset consisted of early apoptotic cells with fragmented chromatin, and could be contaminated with nuclei expelled from erythroblasts. Concluding, murine bone marrow Lin − Sca-1 + CD45 − FSC low cells are heterogeneous population, which do not express the pluripotency marker Oct-4A. Despite expression of some hematopoietic markers by a Lin − Sca-1 + CD45 − c-Kit + KDR − subset of VSELs, they do not display hematopoietic potential in a clonogenic assay and are enriched in early apoptotic cells.

While intrinsic changes in aging hematopoietic stem cells (HSCs) are well characterized, it remains unclear how extrinsic factors affect HSC aging. Here, we demonstrate that cells in the niche—endothelial cells (ECs) and CXCL12‐abundant reticular cells (CARs)—highly express the heme‐degrading enzyme, heme oxygenase 1 (HO‐1), but then decrease its expression with age. HO‐1‐deficient animals (HO‐1 ^−/− ) have altered numbers of ECs and CARs that produce less hematopoietic factors. HSCs co‐cultured in vitro with HO‐1 ^−/− mesenchymal stromal cells expand, but have altered kinetic of growth and differentiation of derived colonies. HSCs from young HO‐1 ^−/− animals have reduced quiescence and regenerative potential. Young HO‐1 ^−/− HSCs exhibit features of premature exhaustion on the transcriptional and functional level. HO‐1 ^+/+ HSCs transplanted into HO‐1 ^−/− recipients exhaust their regenerative potential early and do not reconstitute secondary recipients. In turn, transplantation of HO‐1 ^−/− HSCs to the HO‐1 ^+/+ recipients recovers the regenerative potential of HO‐1 ^−/− HSCs and reverses their transcriptional alterations. Thus, HSC‐extrinsic activity of HO‐1 prevents HSCs from premature exhaustion and may restore the function of aged HSCs.

Subcutaneous injection of the tumor cell suspension is a simple and commonly used tool for studying tumor development in vivo. However, subcutaneous models poorly resemble tumor complexity due to the fast growth not reflecting the natural course. Here, we describe an application of the new spheroid-plug model to combine the simplicity of subcutaneous injection with improved resemblance to natural tumor progression. Spheroid-plug model relies on in vitro formation of tumor spheroids, followed by injection of single tumor spheroid subcutaneously in Matrigel matrix. In spheroid-plug model, tumors grow slower in comparison to tumors formed by injection of cell suspension as assessed by 3D ultrasonography (USG) and in vivo bioluminescence measurements. The slower tumor growth rate in spheroid-plug model is accompanied by reduced necrosis. The spheroid-plug model ensures increased and more stable vascularization of tumor than classical subcutaneous tumor model as demonstrated by 3D USG Power Doppler examination. Flow cytometry analysis showed that tumors formed from spheroids have enhanced infiltration of endothelial cells as well as hematopoietic and progenitor cells with stem cell phenotype (c-Kit⁺ and Sca-1⁺). They also contain more tumor cells expressing cancer stem cell marker CXCR4. Here, we show that spheroid-plug model allows investigating efficiency of anticancer drugs. Treatment of spheroid-plug tumors with known antiangiogenic agent axitinib decreased their size and viability. The antiangiogenic activity of axitinib was higher in spheroid-plug model than in classical model. Our results indicate that spheroid-plug model imitates natural tumor growth and can become a valuable tool for cancer research.

Granulocyte colony‐stimulating factor (G‐CSF) is used in clinical practice to mobilize cells from the bone marrow to the blood; however, it is not always effective. We show that cobalt protoporphyrin IX (CoPP) increases plasma concentrations of G‐CSF, IL‐6, and MCP‐1 in mice, triggering the mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC). Compared with recombinant G‐CSF, CoPP mobilizes higher number of HSPC and mature granulocytes. In contrast to G‐CSF, CoPP does not increase the number of circulating T cells. Transplantation of CoPP‐mobilized peripheral blood mononuclear cells (PBMC) results in higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G‐CSF. Although CoPP is used to activate Nrf2/HO‐1 axis, the observed effects are Nrf2/HO‐1 independent. Concluding, CoPP increases expression of mobilization‐related cytokines and has superior mobilizing efficiency compared with recombinant G‐CSF. This observation could lead to the development of new strategies for the treatment of neutropenia and HSPC transplantation.

Melanoma-initiating cells (MICs) contribute to the tumorigenicity and heterogeneity of melanoma. MICs are identified by surface and functional markers and have been shown to display cancer stem cell (CSC) properties. However, the existence of MICs that follow the hierarchical CSC model has been questioned by studies showing that single unselected melanoma cells are highly tumorigenic in xenotransplantation assays. Herein, we characterize cells expressing MIC markers (CD20, CD24, CD133, Sca-1, ABCB1, ABCB5, ALDH^high) in the B16-F10 murine melanoma cell line. We use flow cytometric phenotyping, single-cell sorting followed by in vitro clonogenic assays, and syngeneic in vivo serial transplantation assays to demonstrate that the expression of MIC markers does not select CSC-like cells in this cell line. Previously, our group showed that heme-degrading enzyme heme oxygenase-1 (HO-1) can be upregulated in melanoma and increase its aggressiveness. Here, we show that HO-1 activity is important for non-adherent growth of melanoma and HO-1 overexpression enhances the vasculogenic mimicry potential, which can be considered protumorigenic activity. However, HO-1 overexpression decreases clone formation in vitro and serial tumor initiation in vivo. Thus, HO-1 plays a dual role in melanoma, improving the progression of growing tumors but reducing the risk of melanoma initiation.