Katarzyna Mierzejewska

Very small embryonic-like stem cells (VSELs) represent a real challenge in stem cell biology: recent pros and cons in the midst of a lively debate.

The concept that adult tissue, including bone marrow (BM), contains early-development cells with broader differentiation potential has again been recently challenged. In response, we would like to review the accumulated evidence from several independent laboratories that adult tissues, including BM, harbor a population of very rare stem cells that may cross germ layers in their differentiation potential. Thus, the BM stem cell compartment hierarchy needs to be revisited. These dormant, early-development cells that our group described as very small embryonic-like stem cells (VSELs) most likely overlap with similar populations of stem cells that have been identified in adult tissues by other investigators as the result of various experimental strategies and have been given various names. As reported, murine VSELs have some pluripotent stem cell characteristics. Moreover, they display several epiblast/germline markers that suggest their embryonic origin and developmental deposition in adult BM. Moreover, at the molecular level, changes in expression of parentally imprinted genes (for example, Igf2–H19) and resistance to insulin/insulin-like growth factor signaling (IIS) regulates their quiescent state in adult tissues. In several emergency situations related to organ damage, VSELs can be activated and mobilized into peripheral blood, and in appropriate animal models they contribute to tissue organ/regeneration. Interestingly, their number correlates with lifespan in mice, and they may also be involved in some malignancies. VSELs have been successfully isolated in several laboratories; however, some investigators experience problems with their isolation.

Novel evidence that crosstalk between the complement, coagulation and fibrinolysis proteolytic cascades is involved in mobilization of hematopoietic stem/progenitor cells (HSPCs).

The role of blood proteinases in the mobilization of hematopoietic stem/progenitor cells (HSPCs) is still not well understood. As previously reported, activation of the complement cascade (ComC) and cleavage of C5 by C5 convertase are enabling events in the release of C5a that plays a crucial role in the egress of HSPCs from bone marrow (BM) into peripheral blood (PB) and explains why C5-deficient mice are poor mobilizers. Here we provide evidence that during granulocyte colony-stimulating factor- and AMD3100-induced mobilization, not only the ComC but also two other evolutionarily ancient proteolytic enzyme cascades, the coagulation cascade (CoaC) and the fibrynolytic cascade (FibC), become activated. Activation of all three cascades was measured by generation of C5a, decrease in prothrombin time and activated partial thromboplastin time as well as an increase in the concentrations of plasmin/antiplasmin and thrombin/antithrombin. More importantly, the CoaC and FibC, by generating thrombin and plasmin, respectively, provide C5 convertase activity, explaining why mobilization of HSPCs in C3-deficient mice, which do not generate ComC-generated C5a convertase, is not impaired. Our observations shed more light on how the CoaC and FibC modulate stem cell mobilization and may lead to the development of more efficient mobilization strategies in poor mobilizers. Furthermore, as it is known that all these cascades are activated in all the situations in which HSPCs are mobilized from BM into PB (for example, infections, tissue/organ damage or strenuous exercise) and show a circadian rhythm of activation, they must be involved in both stress-induced and circadian changes in HSPC trafficking in PB.

Hematopoietic Stem/Progenitor Cells Express Several Functional Sex Hormone Receptors—Novel Evidence for a Potential Developmental Link Between Hematopoiesis and Primordial Germ Cells

Evidence has accumulated that hematopoietic stem progenitor cells (HSPCs) share several markers with the germline, a connection supported by reports that prolactin, androgens, and estrogens stimulate hematopoiesis. To address this issue more directly, we tested the expression of receptors for pituitary-derived hormones, such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH), on purified murine bone marrow (BM) cells enriched for HSPCs and tested the functionality of these receptors in ex vivo signal transduction studies and in vitro clonogenic assays. We also tested whether administration of pituitary- and gonad-derived sex hormones (SexHs) increases incorporation of bromodeoxyuridine (BrdU) into HSPCs and expansion of hematopoietic clonogenic progenitors in mice and promotes recovery of blood counts in sublethally irradiated animals. We report for the first time that HSPCs express functional FSH and LH receptors and that both proliferate in vivo and in vitro in response to stimulation by pituitary SexHs. Furthermore, based on our observations that at least some of CD45(-) very small embryonic-like stem cells (VSELs) may become specified into CD45(+) HSPCs, we also evaluated the expression of pituitary and gonadal SexHs receptors on these cells and tested whether these quiescent cells may expand in vivo in response to SexHs administration. We found that VSELs express SexHs receptors and respond in vivo to SexHs stimulation, as evidenced by BrdU accumulation. Since at least some VSELs share several markers characteristic of migrating primordial germ cells and can be specified into HSPCs, this observation sheds new light on the BM stem cell hierarchy.

Endurance Exercise Mobilizes Developmentally Early Stem Cells into Peripheral Blood and Increases Their Number in Bone Marrow: Implications for Tissue Regeneration.

Endurance exercise has been reported to increase the number of circulating hematopoietic stem/progenitor cells (HSPCs) in peripheral blood (PB) as well as in bone marrow (BM). We therefore became interested in whether endurance exercise has the same effect on very small embryonic-like stem cells (VSELs), which have been described as a population of developmentally early stem cells residing in BM. Mice were run daily for 1 hour on a treadmill for periods of 5 days or 5 weeks. Human volunteers had trained in long-distance running for one year, six times per week. FACS-based analyses and RT-PCR of murine and human VSELs and HSPCs from collected bone marrow and peripheral blood were performed. We observed that endurance exercise increased the number of VSELs circulating in PB and residing in BM. In parallel, we observed an increase in the number of HSPCs. These observations were subsequently confirmed in young athletes, who showed an increase in circulating VSELs and HSPCs after intensive running exercise. We provide for the first time evidence that endurance exercise may have beneficial effects on the expansion of developmentally early stem cells. We hypothesize that these circulating stem cells are involved in repairing minor exercise-related tissue and organ injuries.

Genome-wide analysis of murine bone marrow‑derived very small embryonic-like stem cells reveals that mitogenic growth factor signaling pathways play a crucial role in the quiescence and ageing of these cells

It has been postulated that the most primitive population of stem cells, Oct4+Sca-1+Lin−CD45− very small embryonic-like stem cells (VSELs), differentiate into tissue-committed stem cells in adult mice. However, Oct4+ VSELs remain quiescent in adult tissues and do not form teratomas. In thi study, we report the characteristics of the VSEL transcriptome by gene set enrichment analysis employing a microarray database established from 20 murine bone marrow-derived, FACS-sorted VSELs in comparison with hematopoietic stem cells and embryonic stem cells. In the Oct4+ VSELs, we observed the upregulation of tissue-specific gene sets and a gene set encoding the complement-coagulation cascade. By contrast, in the VSELs, we observed the downregulation of genes involved in the UV radiation response, mRNA processing and mitogenic growth factor signaling [e.g., insulin-like growth factor-1 (IGF-1) and neurotrophic tyrosine kinase receptor A (TRKA), as well as the ERK and PI3K pathways]. Employing leading-edge subset analysis and real-time PCR assays, we observed that several genes, such as growth factor receptor-bound protein 2 (GRB2), son of sevenless homolog 1 (SOS1), SHC (Src homology 2 domain containing) transforming protein 1 (SHC1), mitogen-activated protein kinase kinase 1 (MAP2K1), v-akt murine thymoma viral oncogene homolog 3 (AKT3), ELK1, ribosomal protein S6 kinase, 90kDa, polypeptide 3 (RPS6KA3), glycogen synthase kinase 3β (GSK3β) and casein kinase 2, alpha 1 polypeptide (CSNK2A1), which are involved in mitogenic growth factor signaling pathways, were commonly downregulated in the VSELs. Notably, this repression was reversed in the VSELs co-cultured over a C2C12 supportive cell-line, whereby they are induced to form VSEL-derived spheres (VSEL-DSs); thus, they are enriched, forming more differentiated stem cells. Therefore, we suggest that the repression of mitogenic growth factor signaling (e.g., through the IGF-1 receptor) may prevent uncontrolled Oct4+ VSEL proliferation and teratoma formation. Thus, restoring the responsiveness to mitogenic growth factors may be a crucial step in employing these cells in regenerative medicine.

A novel view of paroxysmal nocturnal hemoglobinuria pathogenesis: more motile PNH hematopoietic stem/progenitor cells displace normal HSPCs from their niches in bone marrow due to defective adhesion, enhanced migration and mobilization in response to erythrocyte-released sphingosine-1 phosphate gradient

A novel view of paroxysmal nocturnal hemoglobinuria pathogenesis: more motile PNH hematopoietic stem/progenitor cells displace normal HSPCs from their niches in bone marrow due to defective adhesion, enhanced migration and mobilization in response to erythrocyte-released sphingosine-1 phosphate gradient

Evidence that the population of quiescent bone marrow-residing very small embryonic/epiblast-like stem cells (VSELs) expands in response to neurotoxic treatment

The concept that bone marrow (BM)‐derived cells may participate in neural regeneration remains controversial, and the identity of the specific cell type(s) involved remains unknown. We recently reported that the adult murine BM contains a highly mobile population of Sca‐1+Lin−CD45− cells known as very small embryonic/epiblast‐like stem cells (VSELs) that express several markers of pluripotency such as Oct‐4. In the BM microenvironment, these cells are kept quiescent because of epigenetic modification of certain paternally imprinted genes. However, as reported, these cells can be mobilized in mice in an experimental model of stroke and express several genes involved in neurogenesis while circulating in peripheral blood (PB). In the current work, we employed a model of toxic brain damage, which is induced by administration of kainic acid, to see not only whether VSELs can be mobilized into PB in response to this neurotoxin, but, more importantly, whether they proliferate and expand in BM tissue. We report here for the first time that brain damage leads to activation and expansion of the BM pool of quiescent VSELs, which precedes their subsequent egress into PB. Harnessing these cells in neural tissue regeneration is currently one of the challenges in regenerative medicine.

The Effects of Cadmium at Low Environmental Concentrations on THP-1 Macrophage Apoptosis.

Cadmium at environmental concentrations is a risk factor for many diseases, including cardiovascular and neurodegenerative diseases, in which macrophages play an important role. The aim of this study was to evaluate the effects of cadmium at low environmental (nanomolar) concentrations on apoptotic processes in THP-1(acute monocytic leukemia cells line)-derived macrophages, with special focus on mitochondrial events involved. Macrophages were incubated with various cadmium chloride (CdCl2) solutions for 48 h at final concentrations of 5 nM, 20 nM, 200 nM and 2 µM CdCl2. Cell viability was measured using flow cytometry. Flow cytometric measurement (annexin V/FITC (annexin V/fluorescein isothiocyanate) and PI (propidium iodide) double staining) was used to quantify the extent of apoptosis. Fluorescence and confocal microscopy were used for imaging of apoptosis process. Changes in mitochondrial membrane potential were monitored using cytofluorimetry after cell staining with JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyane iodide) probe. Mitochondrial ROS (reactive oxygen species) levels were measured cytofluorimetrically after incubation of cells with mitochondrial superoxide indicator (MitoSOX) red fluorescent marker. The mRNA expression of Bcl-2 and Bax was analysed with qRT-PCR. Our study demonstrates that cadmium, even at low environmental concentrations, exerts mitochondrial toxicity in THP-1 macrophages. Forty-eight-hour exposure to very low concentrations reduces cell viability and results in cell death by apoptosis and necrosis. The decrease in mitochondrial membrane potential, increased ROS production, increased Bax and decreased Bcl-2 mRNA expression are mitochondrial events involved in cadmium-induced apoptosis.

Abstract 4072: Embryonic rest hypothesis of cancer development revisited: functional gonadotropic hormone receptors are expressed by normal and malignant hematopoietic cells and functional erythropoietin receptor is expressed by germline-derived tumors

Background: The existence in adult tissues of developmentally early stem cells with broader specification potential may suggest the presence of embryonic primordial germ cell (PGC) remnants in post-natal organs. To support this small, quiescent stem cells (VSELs) that express several markers of PGCs reside in adult murine bone marrow (BM) (Leukemia 2010;24:1450), and like PGCs, are kept quiescent by erasure of imprinting on paternally imprinted genes (Leukemia 2009;23:2042). As reported hematopoietic stem/progenitor cells (HSPCs) can become specified from a population of migrating PGCs isolated from embryos (Blood 1995;86:463) as well as from adult bone marrow VSELs (Leukemia 2011;25,1278). In support of this intriguing possibility, HSPCs and PGCs are both highly migratory populations of stem cells, and specification of the first primitive HSPCs in yolk sac blood islands as well as the origin of definitive HSPCs in the aorta-gonado-mesonephros region are chronologically and anatomically correlated with the developmental migration of PGCs in extra- and intra-embryonic tissues on their way to the genital ridges. Hypothesis: Based on these observations, we have hypothesized that PGC-derived cells as well as HSPCs share some common receptors, and we tested the expression of gonadotropic hormone receptors (GHR) and erythropoietin receptor (EpoR) on HSPCs and PGC-derived cells, respectively. Materials and Methods. We employed RT-PCR studies to evaluate the expression of GHR on normal and malignant HSPCs, whereas we evaluated the expression of EpoR on teratocarcinoma and ovarian cancer cell lines. The functionality of these receptors was tested by chemotaxis, adhesion, and proliferation assays, and we performed signal transduction studies employing specific ligands for gonadal receptors to stimulate HSPCs and erythropoietin (EPO) to stimulate germline-derived cells. Results. We observed the expression of functional FSH, LH, PRL, estrogen, and androgen receptors on normal murine and human HSPCs and in leukemia cell lines. At the same time, we observed the presence of functional EpoRs on murine and human teratocarcinoma cells and ovarian cancer cell lines. Conclusions. Our data provide further evidence for the existence of a developmental link between germline and hematopoiesis and shed new light on the developmental hierarchy of the stem cell compartment in adult tissues and possibility that some malignancies may develop from embryonic remnants. These observations also have important practical implications: i) pituitary gonadal hormones could be employed in selected cases of BM failure to stimulate hematopoiesis and ii) EPO treatment (e.g., because of anemia after chemotherapy) should be avoided in patients with germline malignancies. Citation Format: Malwina Suszynska, Katarzyna Mierzejewska, Agata Poniewierska-Baran, Ahmed Abdelbasit Ismail, Gabriela Schneider, Pranesh Gunjal, Janina Ratajczak, Sham S. Kakar, Magda Kucia, Mariusz Z. Ratajczak. Embryonic rest hypothesis of cancer development revisited: functional gonadotropic hormone receptors are expressed by normal and malignant hematopoietic cells and functional erythropoietin receptor is expressed by germline-derived tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4072. doi:10.1158/1538-7445.AM2015-4072