Haematopoietic stem cell niche in Drosophila

Development and homeostasis of the haematopoietic system is dependent upon stem cells that have the unique ability to both self-renew and to differentiate in all cell lineages of the blood. The crucial decision between haematopoietic stem cell (HSC) self-renewal and differentiation must be tightly controlled. Ultimately, this choice is regulated by the integration of intrinsic signals together with extrinsic cues provided by an exclusive microenvironment, the so-called haematopoietic niche. Although the haematopoietic system of vertebrates has been studied extensively for many decades, the specification of the HSC niche and its signals involved are poorly understood. Much of our current knowledge of how niches regulate long-term maintenance of stem cells is derived from studies on Drosophila germ cells. Now, two recently published studies by Mandal et al.1 and Krezmien et al.2 describe the Drosophila haematopoietic niche and signal transduction pathways that are involved in the maintenance of haematopoietic precursors. Both reports emphasize several features that are important for controlling stem cell behavior and show parallels to both the vertebrate haematopoietic niche as well as the Drosophila germline stem cell niches in ovary and testis. The findings of both papers shed new light on the specific interactions between haematopoietic progenitors and their microenvironment.     

Insights into the biology of cord blood stem/progenitor cells

OBJECTIVES: To review information on cord blood banking and transplantation with respect to the author's studies, and in context of this field of investigation. RESULTS: Cord blood transplantation has been successfully used to treat a number of malignant and non-malignant disorders. However, this technique is still associated with limited numbers of cells for transplantation, and with delayed engraftment of neutrophils and platelets. The field of cord blood transplantation will benefit from enhanced and mechanistically based information on haematopoietic stem cell function and potential means to enhance its effectiveness are reviewed. This includes notions concerning possibility of retrieving more cells from the placenta and cord blood, to expand haematopoietic stem cells ex vivo and to increase efficiency of homing and engraftment of these cells. Also discussed are cryopreservation and long-term storage of cord blood haematopoietic and progenitor cells, and new laboratory findings and animal studies for non-haematopoietic uses of cord blood.     

Inflammatory modulation of HSCs: viewing the HSC as a foundation for the immune response

Cells of the innate and adaptive immune systems are the progeny of a variety of haematopoietic precursors, the most primitive of which is the haematopoietic stem cell. Haematopoietic stem cells have been thought of generally as dormant cells that are only called upon to divide under extreme conditions, such as bone marrow ablation through radiation or chemotherapy. However, recent studies suggest that haematopoietic stem cells respond directly and immediately to infections and inflammatory signals. In this Review, we summarize the current literature regarding the effects of infection on haematopoietic stem cell function and how these effects may have a pivotal role in directing the immune response from the bone marrow.     

Recapitulative haematopoietic development of human pluripotent stem cells in the absence of exogenous haematopoietic cytokines

To improve the recapitulative quality of human pluripotent stem cell (hPSC) differentiation, we removed exogenous haematopoietic cytokines from the defined differentiation system. Here, we show that endogenous stimuli and VEGF are sufficient to induce robust hPSC-derived haematopoiesis, intensive generation of haematopoietic progenitors, maturation of blood cells and the emergence of definitive precursor cells including those that phenotypically identical to early human embryonic haematopoietic stem cells (HSCs). Moreover, the cytokine-free system produces significantly higher numbers of haematopoietic progenitors compared to the published protocols. The removal of cytokines revealed a broad developmental potential of the early blood cells, stabilized the hPSC-derived definitive precursors and led to spontaneous activation of inflammatory signalling. Our cytokine-free protocol is simple, efficient, reproducible and applicable for embryonic stem cells (ESCs) and induced PSCs. The spectrum of recapitulative features of the novel protocol makes the cytokine-free differentiation a preferred model for studying the early human haematopoietic development.     

Parathyroid Hormone Administration Improves Bone Marrow Microenvironment and Partially Rescues Haematopoietic Defects in Bmi1-Null Mice

The epigenetic regulator Bmi1 is key in haematopoietic stem cells, and its inactivation leads to defects in haematopoiesis. Parathyroid hormone (PTH), an important modulator of bone homeostasis, also regulates haematopoiesis, so we asked whether PTH administration improves bone marrow microenvironment and rescues the haematopoietic defects in Bmi1-null mice. The mice were treated with PTH1-34 (containing the first 34 residues of mature PTH), an anabolic drug currently used for treating osteoporosis, and compared with the vehicle-treated Bmi1 ^-/- and wild-type littermates in terms of skeletal and haematopoietic phenotypes. We found that the administration significantly increased all parameters related to osteoblastic bone formation and significantly reduced the adipocyte number and PPARγ expression. The bone marrow cellularity, numbers of haematopoietic progenitors and stem cells in the femur, and numbers of lymphocytes and other white blood cells in the peripheral blood all increased significantly when compared to vehicle-treated Bmi1^-/- mice. Moreover, the number of Jagged1-positive cells and percentage of Notch intracellular domain-positive bone marrow cells and protein expression levels of Jagged1 and NICD in bone tissue were also increased in Bmi1 ^-/- mice upon PTH1-34 administration,whereas the up-regulation of PTH on both Notch1 and Jagged1 gene expression was blocked by the Notch inhibitor DAPT administration. These results thus indicate that PTH administration activates the notch pathway and partially rescues haematopoietic defects in Bmi1-null mice, further suggesting that haematopoietic defects in the animals are not only a result of reduced self-renewal of haematopoietic stem cells but also due to impaired bone marrow microenvironment.     

Circulating CXCR4-positive stem/progenitor cells compete for SDF-1-positive niches in bone marrow,muscle and neural tissues: an alternative hypothesis to stem cell plasticity

The trans-differentiation hypothesis of adult tissue-specific stem cells has been recently questioned because of insufficient proof that the so-called plasticity experiments were performed on pure populations of tissue-specific stem cells. It was shown recently, for example, that the formation of haematopoietic colonies by muscle cells depended on the presence of haematopoietic stem/progenitor cells residing within the muscle tissue and hence was not related to the plasticity of the muscle stem cells. The explanation for the presence in, or homing into, muscles of haematopoietic stem cells is, however, not clear. In our study, we hypothesised that muscle tissues secrete stromal-derived factor (SDF)- 1, an alpha-chemokine for haematopoietic stem cells (HSC), which could attract HSC circulating in peripheral blood into muscle tissue. We found, using RT-PCR and immunocytochemistry, that SDF-1 was expressed in human heart and skeletal muscles. Moreover, muscle satellite cells, which are pivotal for regeneration of muscle, highly expressed on their surface CXCR4, a G-protein-coupled receptor that binds SDF-1. To determine whether the CXCR4 receptor is functional on muscle satellite/progenitor cells, we stimulated murine satellite cells (the C2C12 cell line) with SDF-1 and demonstrated the phosphorylation of p42/44 MAPK and AKT serine-threonine kinase in these cells. Moreover, we showed that SDF-1 gradient chemoattracts these cells. We postulate that the CXCR4-positive muscle satellite and CXCR4-positive HSC circulating in the peripheral blood compete for occupancy of SDF-1-positive stem cell niches that are present in bone marrow and muscle tissues. Thus, we suggest that competition for common niches by various circulating CXCR4-positive stem cells and their ability to home to the SDF-1-positive niches in various organs, is a better explanation than stem cell plasticity of why (i) haematopoietic colonies can be cultured from muscles and (ii) early muscle progenitors could be cultured from bone marrow.     

The transplantability of bone marrow and spleen cells after filtration through silon tissue]

Investigations were carried out on the separation of haematopoietic stem cells from suspensions of the bone-marrow and spleen by means of filtration with silon tissue. The presence of stem cells in the filtrates was determined by the spleen colony test according to the method of Till and McCulloch in irradiated mice. The investigations revealed that a selective separation of haematopoietic stem cells could not be achieved when proceeding in this way. From the results of further test series, in which suspensions were also used which had been gained from haematopoietic tissues of hypersplenic mice, the conclusion could be drawn that the haematopoietic stem cells obtained by filtrating the bone-marrow will have another affinity to the spleen tissue of irradiated mice than the haematopoietic stem cells gained by filtrating the spleen tissue.     

Effect of stem cell mobilization with cyclophosphamide plus granulocyte colony-stimulating factor on morphology of haematopoietic organs in mice

Both granulocyte colony-stimulating factor (G-CSF) and cyclophosphamide (CY) are employed in the clinic as mobilizing agents to stimulate the egress of haematopoietic stem/progenitor cells (HSPC) from bone marrow (BM) into peripheral blood (PB). However, although both compounds are effective, the simultaneous administration of G-CSF + CY allows for optimal mobilization. The aim of this study was to compare morphological changes in major haematopoietic organs in mice mobilized by G-CSF + CY. We employed the standard G-CSF + CY mobilization protocol, in which mice were injected at day 0 with a single dose of CY followed by daily injection of G-CSF for 6 consecutive days. We noticed that the cytoreductive effect of CY on BM and spleen tissue was compensated at day 2 by the pro-proliferative effect of G-CSF. Furthermore, as evidenced by histological examination of BM sections at day 4, egress of haematopoietic cells from BM was accelerated by 2 days as compared to mobilization by G-CSF or CY alone; also, by day 6 there was accumulation of early haematopoietic (Thy-l(low) c-kit+) cells in the spleens and livers of mobilized animals. This implies that HSPC that are mobilized from BM and circulate in PB may 'home' to peripheral organs. We envision that such an accumulation of these cells in the spleen (which is a major haematopoietic organ in mouse) allows them to participate in haematopoietic reconstitution. Their homing to other sites (for example the liver) is evidence that BM-derived stem cells are playing a pivotal role in organ/tissue regeneration. The potential involvement of major chemoattractants for stem cells, like stromal-derived factor-1 which is induced by CY in various regenerating organs such as the liver, requires further study. We conclude that inclusion of CY into mobilization protocols on the one hand efficiently increases the egress of HSPC from the BM, but on the other hand may lead to the relocation of BM stem cell pools to peripheral tissues.     

Human haematopoiesis in steady state and following intense perturbations

Haematopoiesis is comprised of multiple stages, originating from pluripotent stem cells through intermediate progenitors to mature differentiated cells. Consequently, during the development of blood cells numerous sites are potentially exposed to the intense perturbations induced by anticancer chemotherapy. However, little is known about human haematopoietic stem cell kinetics in health and following cytotoxic perturbations. Here we reconstruct the complex in vivo dynamics of haematopoietic populations, including the elusive pluripotent stem cells, with a detailed mathematical representation of the marrow biology. The bone marrow kinetic parameters were estimated by using white blood cell counts routinely collected in patients during high dose chemotherapy (HDCT) followed by autologous peripheral blood stem cell transplantation and granulocyte colony stimulating factor (G-CSF) injections. Studying the model performance under a wide variety of parameter values reveals that bone marrow is surprisingly robust in the physiologically feasible parameter space. We infer that the human haematopoietic pluripotent stem cell density is approximately 1 in 2 · 10⁵ mononuclear cells and that most of these cells are quiescent, dividing once in 3–4 weeks. Our results suggest that the re-infused stem cell content is relatively high (10⁴ kg⁻¹ or 1/300 of CD34+ cells) which contributes to both the long-term marrow re-population as well as to short-term support. This study implies that, in most patients, the pluripotent population recovers within 4 months following HDCT. The proposed model accurately predicts the bone marrow dynamics over a wide range of perturbations caused by clinical interventions. It provides valuable insights about the haematopoietic regeneration capacity, predicts the effect of G-CSF manipulation and of ex vivo graft expansion in improving transplantation procedures, and may have implications for effective stem cell gene therapy.     

Identification of DNA methylation changes at cis-regulatory elements during early steps of HSC differentiation using tagmentation-based whole genome bisulfite sequencing

Epigenetic alterations during cellular differentiation are a key molecular mechanism which both instructs and reinforces the process of lineage commitment. Within the haematopoietic system, progressive changes in the DNA methylome of haematopoietic stem cells (HSCs) are essential for the effective production of mature blood cells. Inhibition or loss of function of the cellular DNA methylation machinery has been shown to lead to a severe perturbation in blood production and is also an important driver of malignant transformation. HSCs constitute a very rare cell population in the bone marrow, capable of life-long self-renewal and multi-lineage differentiation. The low abundance of HSCs has been a major technological barrier to the global analysis of the CpG methylation status within both HSCs and their immediate progeny, the multipotent progenitors (MPPs). Within this Extra View article, we review the current understanding of how the DNA methylome regulates normal and malignant hematopoiesis. We also discuss the current methodologies that are available for interrogating the DNA methylation status of HSCs and MPPs and describe a new data set that was generated using tagmentation-based whole genome bisulfite sequencing (TWGBS) in order to comprehensively map methylated cytosines using the limited amount of genomic DNA that can be harvested from rare cell populations. Extended analysis of this data set clearly demonstrates the added value of genome-wide sequencing of methylated cytosines and identifies novel important cis-acting regulatory regions that are dynamically remodeled during the first steps of haematopoietic differentiation.     

Evaluation of neural plasticity in adult stem cells

The role of stem cells has long been known in reproductive organs and various tissues including the haematopoietic system and skin. During the last decade, stem cells have also been identified in other organs, including the nervous system, both during development and in post-natal life. More recently, evidence has been presented that stem cells thought to be responsible for the generation of mature differentiated cells of one organ, such as haematopoietic stem cells, may have the ability to also differentiate across lineages and contribute to tissues other than haematopoietic cells, including neuronal tissue, suggesting that easily accessible stem cells sources may one day be useful in the therapy of ischaemic (stroke) and also degenerative diseases of the nervous system. Here, we will evaluate the validity of such claims based on a number of criteria we believe need to be fulfilled to definitively conclude that certain stem cells can give rise to functional neural cells that might be suitable for therapy of neural disorders.     

Dynamic niches in the origination and differentiation of haematopoietic stem cells

Haematopoietic stem cells (HSCs) are multipotent, self-renewing progenitors that generate all mature blood cells. HSC function is tightly controlled to maintain haematopoietic homeostasis, and this regulation relies on specialized cells and factors that constitute the haematopoietic 'niche', or microenvironment. Recent discoveries, aided in part by technological advances in in vivo imaging, have engendered a new appreciation for the dynamic nature of the niche, identifying novel cellular and acellular niche components and uncovering fluctuations in the relative importance of these components over time. These new insights significantly improve our understanding of haematopoiesis and raise fundamental questions about what truly constitutes a stem cell niche.     

The bone marrow at the crossroads of blood and immunity

Progenitor cells that are the basis for all blood cell production share the bone marrow with more mature elements of the adaptive immune system. Specialized niches within the bone marrow guide and, at times, constrain the development of haematopoietic stem and progenitor cells (HSPCs) and lineage-restricted immune progenitor cells. Specific niche components are organized into distinct domains to create a diversified landscape in which specialized cell differentiation or population expansion programmes proceed. Local cues that reflect the tissue and organismal state affect cellular interactions to alter the production of a range of cell types. Here, we review the organization of regulatory elements in the bone marrow and discuss how these elements provide a dynamic means for the host to modulate stem cell and adaptive immune cell responses to physiological challenges.     

Therapy targets in glioblastoma and cancer stem cells: lessons from haematopoietic neoplasms

Despite intense efforts to identify cancer‐initiating cells in malignant brain tumours, markers linked to the function of these cells have only very recently begun to be uncovered. The notion of cancer stem cell gained prominence, several molecules and signalling pathways becoming relevant for diagnosis and treatment. Whether a substantial fraction or only a tiny minority of cells in a tumor can initiate and perpetuate cancer, is still debated. The paradigm of cancer‐initiating stem cells has initially been developed with respect to blood cancers where chronic conditions such as myeloproliferative neoplasms are due to mutations acquired in a haematopoietic stem cell (HSC), which maintains the normal hierarchy to neoplastic haematopoiesis. In contrast, acute leukaemia transformation of such blood neoplasms appears to derive not only from HSCs but also from committed progenitors that cannot differentiate. This review will focus on putative novel therapy targets represented by markers described to define cancer stem/initiating cells in malignant gliomas, which have been called ‘leukaemia of the brain’, given their rapid migration and evolution. Parallels are drawn with other cancers, especially haematopoietic, given the similar rampant proliferation and treatment resistance of glioblastoma multiforme and secondary acute leukaemias. Genes associated with the malignant conditions and especially expressed in glioma cancer stem cells are intensively searched. Although many such molecules might only coincidentally be expressed in cancer‐initiating cells, some may function in the oncogenic process, and those would be the prime candidates for diagnostic and targeted therapy. For the latter, combination therapies are likely to be envisaged, given the robust and plastic signalling networks supporting malignant proliferation.     

Cancer stem cells: lessons from leukaemia

Increasing evidence suggests that leukaemias are sustained by leukaemic stem cells. Leukaemia can indeed be viewed as aberrant haematopoietic processes initiated by rare leukaemic stem cells that have maintained or re-acquired the capacity for indefinite proliferation through accumulated mutations and/or epigenetic changes. Yet, despite their critical importance, much remains to be learned about the developmental origin of leukaemic stem cells and the molecular pathways underlying the transformation of normal cells into leukaemic stem cells. This report will review our current knowledge on leukaemic stem cells development and finally demonstrate how these discoveries provide a paradigm for identification of cancer stem cells from solid tumours.     

Stem cells: an alternative to organ transplantation in chronic, degenerative and infectious diseases?

Even in the absence of damage or illness mature animals need billions of new cells every single day of their lives in order to survive and renew circulating blood cells and intestinal and skin lining. This task is accomplished by undifferentiated cells residing in most adult organs. These cells are designated adult stem cells (ASC) since they represent the adult counterpart, present in almost every organ, of the embryonal stem cells (ES) from which the entire human body develops. Scientists first hypothesized the existence of stem cells over a century ago, and haematopoietic stem cells (HSC) have been exploited for the therapy of human diseases for two decades. Other types of stem cells also circulating in the bloodstream have been described. We briefly describe the potential uses of each of these types of cells, including autologous circulating stem cells, for disease therapy and in particular for the possible reversal of liver failure due to chronic hepatitis and/or cirrhosis.