Bone Marrow Hematopoietic Niche Research Articles

Canonical Signaling By TGF Family Members in Mesenchymal Stromal Cells Is Dispensable for Hematopoietic Niche Maintenance Under Basal and Stress Conditions

The bone marrow contains a complex population of stromal and hematopoietic cells that together generate a unique microenvironment, or niche, to support hematopoiesis. Mesenchymal stromal cells are an important component of the bone marrow hematopoietic niche and include CXCL12-abundant reticular (CAR) cells, adipocytes, osteolineage cells, and arteriolar pericytes, all of which have been implicated in hematopoietic stem/progenitor cell (HSPC) maintenance. There also is evidence that adaptive changes in bone marrow stromal cells contributes to recovery from myelosuppresive therapy and the development of certain hematopoietic malignancies. However, the signals that contribute to the development, maintenance, and stress response of bone marrow mesenchymal stromal cells are poorly understood. Here, we test the hypothesis that cytokines of the transforming growth factor superfamily, which include bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), and activins/inhibins, provide signals to mesenchymal stromal cells that contribute to basal and stress hematopoiesis responses. To test this hypothesis, we abrogated canonical TGF family signaling in mesenchymal stem/progenitor cells by deleting Smad4 using a doxycycline-repressible Osterix-Cre transgene (Osx-Cre), which targets all mesenchymal stromal cells in the bone marrow. We first performed lineage-tracing studies using Osx-Cre Smad4fl/fl Ai9 mice to show that activation of Osx-Cre at birth (by removal of doxycycline) results in the efficient targeting of bone marrow mesenchymal stromal cells. Moreover, we show that Smad4 mRNA expression is essentially undetectable in sorted mesenchymal stromal cells sorted from the bone marrow of these mice. Basal hematopoiesis and bone marrow stromal cells were analyzed in 6-8 week old Osx-Cre Smad4fl/fl mice. No alterations in the number or spatial organization of CAR cells, osteoblasts, or adipocytes was observed, and expression of key niche factors, including Scf, Cxcl12, and Spp1 was normal. Basal hematopoiesis, including the number of phenotypic HSCs in bone marrow and spleen, also was normal. Recent studies have shown that inhibition of activin signaling by treating with an activin receptor 2 alpha (ACVR2a) ligand trap stimulates erythropoiesis. Although ACVR2a signaling in erythroid progenitors contributes to this effect, two groups showed that inhibition of ACVR2a signaling in bone marrow stromal cells also stimulates erythropoiesis. Thus, we next characterized basal and stress erythropoiesis in Osx-Cre Smad4fl/fl mice. The frequency of phenotypic erythroid progenitors in bone marrow and spleen was similar to control mice. The stress erythropoiesis response was assessed after induction of acute hemolytic anemia by phenylhydrazine treatment. Both the magnitude of anemia and kinetics of erythroid recovery were similar to control mice. Myelosuppressive therapy induces marked alterations in the bone marrow microenvironment that includes an expansion of osteolineage cells and adipocytes, which have been linked to hematopoietic recovery. Thus, we next characterized stress hematopoiesis in Osx-Cre Smad4fl/fl mice in response to 5-fluorouracil (5-FU) treatment. Compared to control mice, the magnitude and duration of neutropenia following 5-FU were similar. Moreover, mouse survival after repeated weekly doses of 5-FU was comparable to control mice. HSPC mobilization by G-CSF is due, in large part, by downregulation of CXCL12 expression in bone marrow mesenchymal stromal cells. A prior study suggested that SMAD signaling negatively regulates CXCL12 expression in stromal cells. Consistent with this finding, we show that treatment of cultured bone marrow derived MSCs with TGF-b1 for 48 hours results in a significant (3.3-fold, P<0.0001) decrease in CXCL12 mRNA expression. Thus, in the final experiments, we characterized G-CSF induced HSPC mobilization in Osx-Cre, Smad4fl/fl or Osx-Cre, Tgfbr2fl/fl mice. HSPC mobilization, as quantified by CFU-C and Kit+ Sca+ lineage- (KSL) cell number in blood or spleen after 5 days of G-CSF treatment was comparable to control mice. Collectively, these data suggest the TGF family member signaling in mesenchymal stromal cells is dispensable for hematopoietic niche maintenance under basal and stress conditions. Disclosures No relevant conflicts of interest to declare.

Inferring Gene Networks in Bone Marrow Hematopoietic Stem Cell-Supporting Stromal Niches Populations

The cardinal property of bone marrow (BM) stromal cells is their capacity to contribute to Hematopoietic Stem Cell (HSC) niches by providing mediators assisting HSC functions. In this study we first contrasted transcriptomes of stromal cells at different developmental stages and then included large number of HSC-supportive and non-supportive samples. Application of a combination of algorithms enabled identifying a gene network characteristic of the BM stromal HSC-supportive capacity and defining niche populations of perivascular cells, osteoblasts and mesenchymal stromal cells. Inclusion of single cell transcriptomes enabled establishing for the perivascular cell subset a partially oriented graph of direct gene-to-gene interactions. As proof of concept we showed that R-spondin-2, expressed by the perivascular subset, synergized with Kit ligand to amplify ex vivo hematopoietic precursors. Our strategy, first defining the gene organization underlying a functional phenotype then identifying the cell sets contributing to that phenotype, may be generalized to any transcriptome analysis.

Aging of the Hematopoietic Stem Cell Niche: An Unnerving Matter.

The drivers of aging in the hematopoietic system remain incompletely understood. In this issue of Cell Stem Cell, Ho etal. (2019) report that functional switching of β-adrenergic nerve signaling underlies remodeling of stem cell niches, driving age-associated alterations in hematopoiesis.

Osteopontin is An Important Regulative Component of the Fetal Bone Marrow Hematopoietic Stem Cell Niche.

Osteopontin (OPN) is an important component in both bone and blood regulation, functioning as a bridge between the two. Previously, thrombin-cleaved osteopontin (trOPN), the dominant form of OPN in adult bone marrow (BM), was demonstrated to be a critical negative regulator of adult hematopoietic stem cells (HSC) via interactions with α4β1 and α9β1 integrins. We now demonstrate OPN is also required for fetal hematopoiesis in maintaining the HSC and progenitor pool in fetal BM. Specifically, we showed that trOPN is highly expressed in fetal BM and its receptors, α4β1 and α9β1 integrins, are both highly expressed and endogenously activated on fetal BM HSC and progenitors. Notably, the endogenous activation of integrins expressed by HSC was attributed to high concentrations of three divalent metal cations, Ca2+, Mg2+ and Mn2+, which were highly prevalent in developing fetal BM. In contrast, minimal levels of OPN were detected in fetal liver, and α4β1 and α9β1 integrins expressed by fetal liver HSC were not in the activated state, thereby permitting the massive expansion of HSC and progenitors required during early fetal hematopoiesis. Consistent with these results, no differences in the number or composition of hematopoietic cells in the liver of fetal OPN-/- mice were detected, but significant increases in the hematopoietic progenitor pool in fetal BM as well as an increase in the BM HSC pool following birth and into adulthood were observed. Together, the data demonstrates OPN is a necessary negative regulator of fetal and neonatal BM progenitors and HSC, and it exhibits preserved regulatory roles during early development, adulthood and ageing.

The Autophagy in Osteoimmonology: Self-Eating, Maintenance, and Beyond.

It has been long realized that the immune and skeletal systems are closely linked. This crosstalk, also known as osteoimmunology, is a primary process required for bone health. For example, the immune system acts as a key regulator in osteoclasts-osteoblasts coupling to maintain the balanced bone remodeling. Osteoimmunology is achieved through many cellular and molecular processes, among which autophagy has recently been found to play an indispensable role. Autophagy is a highly conserved process in eukaryotic cells, by which the cytoplasm components such as dysfunctional organelles are degraded through lysosomes and then returned to the cytosol for reuse. Autophagy is present in all cells at basal levels to maintain homeostasis and to promote cell survival in response to cellular stress conditions such as nutrition deprivation and hypoxia. Autophagy is a required process in immune cell activation/polarization and osteoclast differentiation, which protecting cells from oxidative stress. The essential of autophagy in osteogenesis is its involvement in osteoblast differentiation and mineralization, especially the role of autophagosome in extracellular calcium transportation. The modulatory feature of autophagy in both immune and skeleton systems suggests its crucial roles in osteoimmunology. Furthermore, autophagy also participates in the maintenance of bone marrow hematopoietic stem cell niche. The focus of this review is to highlight the role of autophagy in the immune-skeleton interactions and the effects on bone physiology, as well as the future application in translational research.

Remodeling of Bone Marrow Hematopoietic Stem Cell Niches Promotes Myeloid Cell Expansion during Premature or Physiological Aging.

SummaryHematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes β2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced β3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with β3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.

Decellularized Wharton jelly matrix: a biomimetic scaffold for ex vivo hematopoietic stem cell culture

AbstractHematopoietic stem progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic “niche,” a special 3-dimensional (3D) microenvironment that regulates HSPC self-renewal and multipotency. In this study, we evaluated a novel 3D in vitro culture system that uses components of the BM hematopoietic niche to expand umbilical cord blood (UCB) CD34+ cells. We developed this model using decellularized Wharton jelly matrix (DWJM) as an extracellular matrix (ECM) scaffold and human BM mesenchymal stromal cells (MSCs) as supporting niche cells. To assess the efficacy of this model in expanding CD34+ cells, we analyzed UCB CD34+ cells, following culture in DWJM, for proliferation, viability, self-renewal, multilineage differentiation, and transmigration capability. We found that DWJM significantly expanded UCB HSPC subset. It promoted UCB CD34+ cell quiescence, while maintaining their viability, differentiation potential with megakaryocytic differentiation bias, and clonogenic capacity. DWJM induced an increase in the frequency of c-kit+ cells, a population with enhanced self-renewal ability, and in CXCR4 expression in CD34+ cells, which enhanced their transmigration capability. The presence of BM MSCs in DWJM, however, impaired UCB CD34+ cell transmigration and suppressed CXCR4 expression. Transcriptome analysis indicated that DWJM upregulates a set of genes that are specifically involved in megakaryocytic differentiation, cell mobility, and BM homing. Collectively, our results indicate that the DWJM-based 3D culture system is a novel in vitro model that supports the proliferation of UCB CD34+ cells with enhanced transmigration potential, while maintaining their differentiation potential. Our findings shed light on the interplay between DWJM and BM MSCs in supporting the ex vivo culture of human UCB CD34+ cells for use in clinical transplantation.

Acute Leukemia Induces Senescence and Impaired Osteogenic Differentiation in Mesenchymal Stem Cells Endowing Leukemic Cells with Functional Advantages.

Mesenchymal stem cells (MSC) constitute an important cell population of the bone marrow hematopoietic niche that supports normally hematopoietic stem cells (HSC) but eventually also leukemic cells. The alterations that occur in the MSC under leukemic stress are not well known. To deepen on this topic, we have used an in vitro model of the leukemic niche (LN) by coculturing MSC with an acute lymphocytic leukemia cell line (REH) and proceeded to evaluate MSC characteristics and functions. We found that leukemic cells induced in MSC a significant increase both in senescence-associated β-galactosidase activity and in p53 gene expression. MSC in the LN also showed a persistent production of cytoplasmic reactive oxygen species (ROS) and a G2/M phase arrest of the cell cycle. Another acute leukemic cell line (SUP-B15) produced almost the same effects on MSC. REH cells adhere strongly to MSC possibly as a result of an increased expression of the adhesion molecules VCAM-1, ICAM-1, and CD49e in MSC and of CD49d in REH cells. Although mesensphere formation was normal or even increased, multipotent differentiation capacity was impaired in MSC from the LN. A REH-conditioned medium was only partially (about 50%) capable of inducing the same changes in MSC, suggesting that cell-to-cell contact is more efficient in inducing these changes. Despite these important effects on MSC in the LN, REH cells increased their cell adhesion, proliferation rate, and directed-migration capacity. In conclusion, in this in vitro LN model, leukemic cells affect importantly the MSC, inducing a senescence process that seems to favour leukemic cell growth.

Neurological Regulation of the Bone Marrow Niche.

The bone marrow (BM) hematopoietic niche is the microenvironment where in the adult hematopoietic stem and progenitor cells (HSPCs) are maintained and regulated. This regulation is tightly controlled through direct cell-cell interactions with mesenchymal stromal stem (MSCs) and reticular cells, adipocytes, osteoblasts and endothelial cells, through binding to extracellular matrix molecules and through signaling by cytokines and hematopoietic growth factors. These interactions provide a healthy environment and secure the maintenance of the HSPC pool, their proliferation, differentiation and migration. Recent studies have shown that innervation of the BM and interactions with the peripheral sympathetic neural system are important for maintenance of the hematopoietic niche, through direct interactions with HSCPs or via interactions with other cells of the HSPC microenvironment. Signaling through adrenergic receptors (ARs), opioid receptors (ORs), endocannabinoid receptors (CRs) on HSPCs and MSCs has been shown to play an important role in HSPC homeostasis and mobilization. In addition, a wide range of neuropeptides and neurotransmitters, such as Neuropeptide Y (NPY), Substance P (SP) and Tachykinins, as well as neurotrophins and neuropoietic growth factors have been shown to be involved in regulation of the hematopoietic niche. Here, a comprehensive overview is given of their role and interactions with important cells in the hematopoietic niche, including HSPCs and MSCs, and their effect on HSPC maintenance, regulation and mobilization.

TGF-β Signaling in Fetal Mesenchymal Progenitor Cells Plays an Essential Role in the Emergence of Bone Marrow Hematopoietic Niches

Hematopoietic stem/progenitor cells (HSPC) reside in a unique microenvironment within the bone marrow called the bone marrow hematopoietic niche. Mesenchymal stromal cells, including CXCL12-abundant reticular (CAR) cells, osteoblasts, arteriolar pericytes, and adipocytes are all important components of the niche. The development and maintenance of mesenchymal stromal cells in the bone marrow is not well characterized. A prior study suggested that these stromal cells are derived from two distinct types of mesenchymal stem/progenitor cells (MSPCs). Primitive MSPCs are present in fetal bone and are responsible for osteoblasts, CAR cells, and adipocytes through approximately 3 weeks after birth, and definitive MPSCs are present at birth and generate bone marrow mesenchymal stromal cells in adult mice. In this study, we abrogated transforming growth factor-b (TGF-β) signaling in MSPCs by deleting Tgfbr2in mesenchymal cells using a doxycycline-repressible Sp7(osterix)-Cre transgene (Osx-Cre).We previously reported that loss of TGF-βsignaling during fetal development results in a marked expansion of CAR cells and adipocytes in the bone marrow, while osteoblasts are significantly reduced. These stromal alterations are associated with significant defects in hematopoiesis, including a shift from lymphopoiesis to myelopoiesis. However, hematopoietic stem cell function is preserved. Interestingly, TGF-βsignaling is dispensable for the maintenance of mesenchymal cells in the bone marrow after birth under steady state conditions. These data show that TGF-βplays an essential role in the lineage specification of fetal but not definitive MSPCs and is required for the establishment of normal hematopoietic niches in fetal and perinatal bone marrow.Canonical TGF-bsignaling is dependent on SMAD4. To investigate whether MSPC lineage specification by TGF-bis dependent on SMAD4, we generated Osx-Cre Smad4Δ/Δmice. Osx-Cre Smad4Δ/Δmice are runted to a similar degree as Osx-CreTgfbr2Δ/Δmice secondary to a loss of mature osteoblasts. However, the magnitude of the increase in bone marrow adiposity is significantly reduced in Osx-Cre Smad4∆/∆mice compared to Osx-Cre, Tgfbr2Δ/Δmice. These data suggested that non-canonical signaling contributes to the suppressive effect of TGF-b on adipogenesis. To test this hypothesis, we generated cultures of mesenchymal stromal cells from wildtype neonatal bone marrow. As expected, in wildtype cultures, the addition of TGF-bpotently suppressed adipocyte formation. To assess the role of MAPK activation on the suppression of adipogenesis by TGF-b, we pharmacologically inhibited MAPK activation. Inhibition of MAPK alone did not suppress adipocyte formation. However, it completely blocked the suppressive effect of TGF-bon adipogenesis. Prior studies showed that phosphorylation of serine 82 of PPARgby MAPK decreases its transcriptional activity. Since PPARgis a master regulator of adipogenesis, we assessed the ability of TGF-b to induce PPARgphosphorylation. Indeed, the addition of TGF-b to the MSPC cultures resulted in reproducible PPARgphosphorylation. These data suggest that TGF-b suppresses adipocyte specification of MSPCs, in part, in a MAPK-dependent fashion through phosphorylation of PPARg.In summary, our data suggest that TGF-b plays a key role in the lineage specification of fetal MSPCs during development and is required for the proper development of fetal hematopoietic niches in the bone marrow. The contribution of TGF-b signaling in MSPCs to the stromal and hematopoietic response to different stressors is an active area of investigation. DisclosuresNo relevant conflicts of interest to declare.

Pharmacological Targeting of Osteoblast-Induced MDS and AML

The bone marrow hematopoietic stem cell niche has emerged as a regulator and instigator of hematological myeloid malignancies. Activation of b-catenin signaling in osteoblasts, by deletion of exon3 or by inactivating mutations in APC, induces myelodysplasia (MDS), rapidly progressing to acute myeloid leukemia (AML) in mice. Activated b-catenin signaling is present in osteoblasts of one third of MDS and AML patients and it is the most active pathway in stromal cells of MDS patients suggesting that it may sustain dysplastic hematopoiesis and progression to MDS and AML in humans. Therefore, targeting this pathway may provide a new therapeutic approach for this subgroup of MDS/AML patients. To examine this hypothesis we searched for an FDA-approved compound with the ability to inhibit b-catenin signaling and identified all-trans-retinoic acid (ATRA) as a potential candidate. We show here that treatment of osteoblastic cells with ATRA inhibits the induction of b-catenin target genes by Wnt3a and abrogates nuclear localization of b-catenin, as shown by immunohistochemistry and western blot analysis. These effects are at least in part independent of its phosphorylation by GSK3b on exon3 and are mediated by the retinoic acid receptor a (RARa) since silencing RARa, but not RARb or RARg, in osteoblasts abolishes the inhibitory effect of ATRA on Wnt3a-induced b-catenin signaling. Similarly, treatment of leukemic mice expressing constitutively active b-catenin in their osteoblasts with ATRA, inhibited b-catenin signaling as measured by expression of its target genes Axin2 and Lef1 in bone. At the same time, ATRA improved anemia and thrombocytopenia, decreased the percentage of blasts in bone marrow and blood and prolonged overall survival in these leukemic mice as compared to vehicle-treated mice. The response to ATRA treatment of two MDS patients with active b-catenin in their osteoblasts, and not responding to other treatments, was monitored. The hematologic phenotype of the patients improved and disease status stabilized so that patients remain transfusion independent and disease-related symptom-free with a good quality of life thereafter. The improvement in disease status coincided with absence of b-catenin in the nucleus as flow cytometry analysis showed a high percentage of patient osteoblasts expressing activated b-catenin in pre-therapy samples but not in on-therapy samples. These results suggest that ATRA may find a new use in the treatment of the specific portion of MDS/AML patients with activated b-catenin in their osteoblasts. DisclosuresRaza:Geoptix: Speakers Bureau; Onconova: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Kura Oncology: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Syros: Research Funding.

Distinct roles of mesenchymal stem and progenitor cells during the development of acute myeloid leukemia in mice

AbstractDespite increasing evidence for the involvement of bone marrow (BM) hematopoietic stem cell niche in leukemogenesis, how BM mesenchymal stem and progenitor cells (MSPCs) contribute to leukemia niche formation and progression remains unclear. Using an MLL-AF9 acute myeloid leukemia (AML) mouse model, we demonstrate dynamic alterations of BM cellular niche components, including MSPCs and endothelial cells during AML development and its association with AML engraftment. Primary patient AML cells also induced similar niche alterations in xenografted mice. AML cell infiltration in BM causes an expansion of early B-cell factor 2+ (Ebf2+) MSPCs with reduced Cxcl12 expression and enhanced generation of more differentiated mesenchymal progenitor cells. Importantly, in vivo fate-mapping indicates that Ebf2+ MSPCs participated in AML niche formation. Ebf2+ cell deletion accelerated the AML development. These data suggest that native BM MSPCs may suppress AML. However, they can be remodeled by AML cells to form leukemic niche that might contribute to AML progression. AML induced dysregulation of hematopoietic niche factors like Angptl1, Cxcl12, Kitl, Il6, Nov, and Spp1 in AML BM MSPCs, which was associated with AML engraftment and partially appeared before the massive expansion of AML cells, indicating the possible involvement of the niche factors in AML progression. Our study demonstrates distinct dynamic features and roles of BM MSPCs during AML development.

Adaptive Regulation of Osteopontin Production by Dendritic Cells Through the Bidirectional Interaction With Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) exert immunosuppressive effects on immune cells including dendritic cells (DCs). However, many details of the bidirectional interaction of MSCs with DCs are still unsolved and information on key molecules by which DCs can modulate MSC functions is limited. Here, we report that osteopontin (OPN), a cytokine involved in homeostatic and pathophysiologic responses, is constitutively expressed by DCs and regulated in the DC/MSC cocultures depending on the activation state of MSCs. Resting MSCs promoted OPN production, whereas the production of OPN was suppressed when MSCs were activated by proinflammatory cytokines (i.e., TNF-α, IL-6, and IL-1β). OPN induction required cell-to-cell contact, mediated at least in part, by β1 integrin (CD29). Conversely, activated MSCs inhibited the release of OPN via the production of soluble factors with a major role played by Prostaglandin E2 (PGE2). Accordingly, pretreatment with indomethacin significantly abrogated the MSC-mediated suppression of OPN while the direct addition of exogenous PGE2 inhibited OPN production by DCs. Furthermore, DC-conditioned medium promoted osteogenic differentiation of MSCs with a concomitant inhibition of adipogenesis. These effects were paralleled by the repression of the adipogenic markers PPARγ, adiponectin, and FABP4, and induction of the osteogenic markers alkaline phosphatase, RUNX2, and of the bone-anabolic chemokine CCL5. Notably, blocking OPN activity with RGD peptides or with an antibody against CD29, one of the OPN receptors, prevented the effects of DC-conditioned medium on MSC differentiation and CCL5 induction. Because MSCs have a key role in maintenance of bone marrow (BM) hematopoietic stem cell niche through reciprocal regulation with immune cells, we investigated the possible MSC/DC interaction in human BM by immunohistochemistry. Although DCs (CD1c+) are a small percentage of BM cells, we demonstrated colocalization of CD271+ MSCs with CD1c+ DCs in normal and myelodysplastic BM. OPN reactivity was observed in occasional CD1c+ cells in the proximity of CD271+ MSCs. Altogether, these results candidate OPN as a signal modulated by MSCs according to their activation status and involved in DC regulation of MSC differentiation.

Three-Dimensional Co-culture of Human Hematopoietic Stem/Progenitor Cells and Mesenchymal Stem/Stromal Cells in a Biomimetic Hematopoietic Niche Microenvironment.

The development of cellular therapies to treat hematological malignancies has motivated researchers to investigate ex vivo culture systems capable of expanding the number of hematopoietic stem/progenitor cells (HSPC) before transplantation. The strategies exploited to achieve relevant cell numbers have relied on culture systems that lack biomimetic niche cues thought to be essential to promote HSPC maintenance and proliferation. Although stromal cells adhered to 2-D surfaces can be used to support the expansion of HSPC ex vivo, culture systems aiming to incorporate cell-cell interactions in a more intricate 3-D environment can better contribute to recapitulate the bone marrow (BM) hematopoietic niche in vitro.Herein, we describe the development of a 3-D co-culture system of human umbilical cord blood (UCB)-derived CD34+ cells and BM mesenchymal stem/stromal cell (MSC) spheroids in a microwell-based platform that allows to attain large numbers of spheroids with uniform sizes. Further comparison with a traditional 2-D co-culture system exploiting the supportive features of feeder layers of MSC is provided, while functional in vitro assays to assess the features of HSPC expanded in the 2-D vs. 3-D MSC co-culture systems are suggested.

Application of Stem Cell Technology in Antiaging and Aging-Related Diseases.

Stem cells are one kind of cells that have the potential of proliferation and differentiation. The human beings are originated from a totipotential stem cell-fertilized egg. After birth, the proliferation and differentiation of stem cells contribute to the development and maturation of individual tissues and organs. After maturation, aging is a phases of the life process, the stem cells within the individual's tissues ensure the metabolism of different cells and tissues, such as the hematopoietic stem cells in the bone marrow, which ensure there are still enough red blood cells (RBCs) being responsible for the mission of transporting oxygen after a single RBC has completed its 120-day physiological life cycle. After pathological damage and necrosis occurring on the intestinal epithelial cells or tubular epithelial cells, there will be regenerative epithelial cells continuing to maintain the integrity of the structure and function of the intestine and renal tubules. The role of stem cells in the regeneration and repair of tissues and organs is not only because of the ability of proliferation and differentiation of stem cells but also of the secretion function of stem cells, which secrete various growth factors and cytokines to regulate the tissue microenvironment. For example, mesenchymal stem cells derived from bone marrow are important regulators in bone marrow hematopoietic stem cell niche. Mesenchymal stem cells maintain the "stemness" of hematopoietic stem cells by secreting various cytokines.Aging is a phases of the life process, and all creatures obey this rule of nature. Different organs of the body have different time of entering into aging. Aging is reflected in structural changes and reduced function. Among them, the reduction of regeneration and repair capacity is the main feature of aging. As we age, the aging of stem cells in human tissues is a major cause of the decline in tissue regeneration capacity. Therefore, the elderly's ability of regenerate and repair can be improved by application of advance stem cell technology. It can delay the aging process and treat aged diseases (showed in Fig. 16.1).

Prevention of breast cancer skeletal metastases with parathyroid hormone.

Advanced breast cancer is frequently associated with skeletal metastases and accelerated bone loss. Recombinant parathyroid hormone [teriparatide, PTH(1-34)] is the first anabolic agent approved in the US for treatment of osteoporosis. While signaling through the PTH receptor in the osteoblast lineage regulates bone marrow hematopoietic niches, the effects of anabolic PTH on the skeletal metastatic niche are unknown. Here, we demonstrate, using orthotopic and intratibial models of 4T1 murine and MDA-MB-231 human breast cancer tumors, that anabolic PTH decreases both tumor engraftment and the incidence of spontaneous skeletal metastasis in mice. Microcomputed tomography and histomorphometric analyses revealed that PTH increases bone volume and reduces tumor engraftment and volume. Transwell migration assays with murine and human breast cancer cells revealed that PTH alters the gene expression profile of the metastatic niche, in particular VCAM-1, to inhibit recruitment of cancer cells. While PTH did not affect growth or migration of the primary tumor, it elicited several changes in the tumor gene expression profile resulting in a less metastatic phenotype. In conclusion, PTH treatment in mice alters the bone microenvironment, resulting in decreased cancer cell engraftment, reduced incidence of metastases, preservation of bone microarchitecture and prolonged survival.

Mesenchymal Stromal Cells, Instigator or Suppressor for the Development of MLL-AF9 Induced Acute Myeloid Leukemia?

It has been reported that bone marrow (BM) hematopoietic niche is critical for the maintenance of normal hematopoiesis and the development of hematopoietic disorders. However, little is known about how the different BM niche components contribute to the initiation and progression of acute myeloid leukemia (AML). To explore the role of BM stromal cells in the development of AML, we here characterized the BM stromal cells in AML mouse model induced by transplantation of mouse BM cellstransduced with MLL-AF9 oncogene. We subdivided the BM stromal cells (CD45-TER119-) into mesenchymal stem cells (MSCs, CD31-CD44-CD51+SCA1+), mesenchymal progenitor cells (MPCs, CD31-CD44-CD51+SCA1-), and endothelial cells (CD31+) in the mice after development of AML by multicolor fluorescent activated cell sorting (FACS) (Qian et al., Mol Cell Biol, 2013). We found significantly increased frequencies of BM MSCs (p < 0.0001), MPCs (p < 0.0001), and endothelial cells (p < 0.0001) in the AML mice, indicating an AML-induced alteration of BM stromal cell composition. The increased frequency of MSCs in AML mouse BM was confirmed by the increased number of colony foming unit-fibroblasts in the AML mice (p < 0.0001). Furthermore, in vitro multi-lineage differentiation assay revealed increased adipogenic and osteogenic differentiation capacities of the BM MSCs in the AML mice. Consistent with the increased osteogenic differentiation capacity of AML MSCs, Runx2 mRNA expression was increased in freshly sorted AML MSCs compared to that in the control mice. In addition, quantitative real time PCR on freshly sorted BM MPCs showed a dramatic downregulation of the genes important in hematopoietic stem cell regulation, including angiopoietin like 1 (Angptl1), C-X-C motif chemokine (Cxcl12), and kit ligand (Kitl) in the AML mice. The altered gene expression might contribute to the AML progression.To specifically determine the contribution of MSCs to AML development, we analyzed the MSC population expressing Early B cell factor 2 (Ebf2) (Qian et al., Mol Cell Biol, 2013) by establishing the AML in Ebf2-Egfp transgenic recipient mice. We detected an increased frequency of Ebf2+ cells in the recipient BM after development of AML (p = 0.0018). In addition, by using the triple transgenic Tg(Ebf2-Egfp x Ebf2-CreER x Rosa26-loxpStoploxp-Tomato) mouse model, where Ebf2+ MSCs and their progenies can be traced by tomato fluorescent protein after tamoxifen treatment, we found that the MSCs and MPCs generated from the Ebf2+ MSCs were also increased in the AML mouse BM (p < 0.05).To further investigate the functional contribution of Ebf2+ MSCs to the AML progression, we took advantage of the transgenic Tg(Ebf2-CreER x Rosa26-loxpStoploxp-DTA) mouse model allowing specific deletion of the Ebf2-expressing MSCs. Strikingly, we observed an accelerated initiation of AML, illustrated by the shorter survival of the MSC depleted recipients (Figure 1), compared to the non-depleted recipients, suggesting that Ebf2+ MSCs could suppress AML progression.Taken together, our data show that BM stromal cell composition and function are altered in the mice with AML and the BM MSCs may act to suppress the progression of the MLL-AF9 induced AML. These findings provide new evidence for the niche contribution to the development of AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An unhealthy diet: making sense of a fatty niche for leukemia stem cells

In an era of obesity epidemics and surging cardiovascular mortality, new evidence has linked our unhealthy diets with an unexpected danger—cancer cell survival and resistance to chemotherapy. Leukemia stem cells (LSCs) are the prototypical cancer stem cell, defined by their ability to initiate and maintain leukemia and also to differentiate into mature progeny that lack this self-renewal ability (1). Analogous to hematopoietic stem cells (HSC), their counterparts in normal development, it has been postulated that LSCs also coopt or hijack cell extrinsic support from secreted or cellular factors to support their own survival within the bone marrow and other hematopoietic niches (2,3).

Research progress of B lymphocyte homeostasis after allogeneic hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation(allo-HSCT) is an effective way to treat hematologic diseases. Rapid immune reconstruction after allo-HSCT can reduce the risk of infection, relapse and secondary tumors. B lymphocyte homeostasis plays a very important role in immune reconstruction after allo-HSCT. The B cell activating factor belong to the tumor necrosis factor family(BAFF), chronic graft versus host disease(cGVHD), bone marrow hematopoietic niche and T lymphocyte are main influence factors of B lymphocyte homeostasis. The author summarizes the research progress of B lymphocyte homeostasis after allo-HSCT. Key words: Hematopoietic stem cell transplantation; B-lymphocyte; B-cell activating factor; Graft vs host disease

Physiologic Hematopoiesis Is Dependent on Stromal Expression of Laminin-γ1

OBJECTIVE: The microenvironment of the bone marrow hematopoietic niche includes: 1) blood vessels; 2) adjacent stromal cells; 3) hematopoietic cells; and 4) cytokines, growth factors, and structural support molecules. Injury to the bone marrow niche affects hematopoietic cells through loss of either physical contact with stromal cells or niche-derived growth factors. Extracellular matrix factors required to maintain the steady-state bone marrow perivascular niche and hematopoiesis have not been fully identified. Here, we examine the role of the extracellular matrix protein laminin-γ1 in adult bone marrow for maintaining the perivascular hematopoietic niche and hematopoiesis.METHODS: A global and inducible laminin-γ1 deficient mouse, hereafter referred to as mutant, was generated in which LAMC1 gene recombination could be monitored by a fluorescent reporter transgene. Tamoxifen was used to induce LAMC1 gene recombination and knock-down of laminin-γ1 protein expression in 8-12 week old mice. Tamoxifen-treated mice lacking the inducible Cre transgene were used as controls for all experiments. Analysis of mutant mice was performed 17-24 days following the first dose of tamoxifen. Bone marrow samples were examined by immunohistochemistry for the presence of laminin-1 protein, and hematopoietic tissues, including bone marrow, peripheral blood, spleen, thymus and lymph node were analyzed by flow cytometry for hematopoietic stem and progenitor cells, and mature hematopoietic cell populations, and by ex vivo hematopoietic progenitor cell colony forming assays. To examine the stromal cell contribution to laminin-γ1 mediated hematopoietic dysfunction, control and mutant mice that had not yet undergone LAMC1 gene recombination were then transplanted with wild type bone marrow. After full hematopoietic reconstitution, chimeric mice were induced with tamoxifen and hematopoietic stem and progenitor cells and mature hematopoietic cell populations were examined.RESULTS: LAMC1 gene recombination in the bone marrow of tamoxifen-treated mutant animals varied between 20-45%. With LAMC1 gene recombination, laminin-protein in the bone marrow is rapidly depleted. Bone marrow blood vessels dilate, and the bone marrow becomes hypocellular. Hematopoietic stem and progenitor cells are reduced in number, as are bone marrow B-cell progenitors, and thymic double-positive T-cells. Erythrocyte and thrombocyte numbers are not changed, nor were NK cell populations, the majority of myeloid cell subtypes, or mature B cells in the bone marrow and blood. CD8+ T-cells were increased within the bone marrow, as were CD11b+, Ly6Clo Ly6G+ myeloid cells in the peripheral blood. Examination of gene recombination within each of these cell types was not consistent with cell-intrinsic mechanism of hematopoietic alterations. Analysis of bone marrow chimeric animals identified loss of stromal cell-produced laminin-γ1 as a significant mediator of these hematopoietic alterations.CONCLUSIONS: The rapid depletion of laminin-γ1 from the bone marrow indicates a high basal turnover rate for the extracellular matrix in the bone marrow. The attendant hematopoietic dysfunction that follows loss of laminin-γ1 production in the bone marrow is specific for immature hematopoietic cell populations, and is dependent on stromal cell production of laminin-γ1. This study identifies laminin-γ1 as a niche-dependent regulator of hematopoietic stem and progenitor cell populations, and suggests that that regulation of its production and degradation may be new targets for the study of the hematopoietic niche. DisclosuresNo relevant conflicts of interest to declare.