An age-progressive platelet differentiation path from hematopoietic stem cells causes exacerbated thrombosis.

Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis.

Acute and endothelial-specific Robo4 deletion affect hematopoietic stem cell trafficking independent of VCAM1.

Hematopoietic stem cell (HSC) trafficking is regulated by a number of complex mechanisms. Among them are the transmembrane protein Robo4 and the vascular cell adhesion molecule, VCAM1. Endothelial VCAM1 is a well-known regulator of hematopoietic cell trafficking, and our previous studies revealed that germline deletion of Robo4 led to impaired HSC trafficking, with an increase in vascular endothelial cell (VEC) numbers and downregulation of VCAM1 protein on sinusoidal VECs. Here, we utilized two Robo4 conditional deletion models in parallel with Robo4 germline knockout mice (R4KO) to evaluate the effects of acute and endothelial cell-specific Robo4 deletion on HSC trafficking. Strikingly similar to the R4KO, the acute deletion of Robo4 resulted in altered HSC distribution between the bone marrow and blood compartments, despite normal numbers of VECs and wild-type levels of VCAM1 cell surface protein on sinusoidal VECs. Additionally, consistent with the R4KO mice, acute loss of Robo4 in the host perturbed long-term engraftment of donor wild-type HSCs and improved HSC mobilization to the peripheral blood. These data demonstrate the significant role that endothelial Robo4 plays in directional HSC trafficking, independent of alterations in VEC numbers and VCAM1 expression.

Megakaryocyte progenitor cell function is enhanced upon aging despite the functional decline of aged hematopoietic stem cells.

Age-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. Although young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared with young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging.

Ubiquitous overexpression of CXCL12 confers radiation protection and enhances mobilization of hematopoietic stem and progenitor cells.

C-X-C motif chemokine ligand 12 (CXCL12; aka SDF1α) is a major regulator of a number of cellular systems, including hematopoiesis, where it influences hematopoietic cell trafficking, proliferation, and survival during homeostasis and upon stress and disease. A variety of constitutive, temporal, ubiquitous, and cell-specific loss-of-function models have documented the functional consequences on hematopoiesis upon deletion of Cxcl12. Here, in contrast to loss-of-function experiments, we implemented a gain-of-function approach by generating a doxycycline-inducible transgenic mouse model that enables spatial and temporal overexpression of Cxcl12. We demonstrated that ubiquitous CXCL12 overexpression led to an increase in multipotent progenitors in the bone marrow and spleen. The CXCL12+ mice displayed reduced reconstitution potential as either donors or recipients in transplantation experiments. Additionally, we discovered that Cxcl12 overexpression improved hematopoietic stem and progenitor cell mobilization into the blood, and conferred radioprotection by promoting quiescence. Thus, this new CXCL12+ mouse model provided new insights into major facets of hematopoiesis and serves as a versatile resource for studying CXCL12 function in a variety of contexts.

Viagra Enables Efficient, Single-Day Hematopoietic Stem Cell Mobilization.

Hematopoietic stem cell (HSC) transplantation is a curative treatment for a variety of blood and immune disorders. Currently available methods to obtain donor HSCs are suboptimal, and the limited supply of donor HSCs hampers the success and availability of HSC transplantation therapies. We recently showed that manipulation of vascular integrity can be employed to induce HSC mobilization from the bone marrow to the blood stream, facilitating non-invasive collection of HSCs. Here, we tested whether FDA-approved vasodilators are capable of mobilizing HSCs. We found that a rapid, 2-h regimen of a single oral dose of Viagra (sildenafil citrate) combined with a single injection of the CXCR4 antagonist AMD3100 leads to efficient HSC mobilization at levels rivaling the standard-of-care 5-day regimen of granulocyte-colony stimulating factor (G-CSF/Filgrastim/Neupogen). Our findings solidify vascular integrity as an essential regulator of HSC trafficking and provide an attractive, single-day regimen for HSC mobilization using already FDA-approved drugs.

Clonal and Quantitative In Vivo Assessment of Hematopoietic Stem Cell Differentiation Reveals Strong Erythroid Potential of Multipotent Cells.

Hematopoiesis is arguably one of the best understood stem cell systems; however, significant challenges remain to reach a consensus understanding of the lineage potential, heterogeneity, and relationships of hematopoietic stem and progenitor cell populations. To gain new insights, we performed quantitative analyses of mature cell production from hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor populations. Assessment of the absolute numbers of mature cell types produced by each progenitor cell revealed a striking erythroid dominance of all myeloid-competent progenitors assessed, accompanied by strong platelet reconstitution. All populations with myeloid potential also produced robust numbers of red blood cells and platelets in vivo. Clonal analysis by single-cell transplantation and by spleen colony assays revealed that a significant fraction of HSCs and multipotent progenitors have multilineage potential at the single-cell level. These new insights prompt an erythroid-focused model of hematopoietic differentiation.

Cryopreserved Ex Vivo-Expanded Allogeneic Myeloid Progenitor Cell Product Protects Neutropenic Mice From a Lethal Fungal Infection.

Severe neutropenia induced by chemotherapy or conditioning for hematopoietic cell transplantation often results in morbidity and mortality due to infection by opportunistic pathogens. A system has been developed to generate ex vivo-expanded mouse myeloid progenitor cells (mMPCs) that produce functional neutrophils in vivo upon transplantation in a pathogen challenge model. It has previously been demonstrated that transplantation of large numbers of freshly isolated myeloid progenitors from a single donor provides survival benefit in radiation-induced neutropenic mice. In the present work, an ex vivo-expanded and cryopreserved mMPC product generated from an allogeneic donor pool retains protective activity in vivo in a lethal fungal infection model. Infusion of the allogeneic pooled mMPC product is effective in preventing death from invasive Aspergillus fumigatus in neutropenic animals, and protection is dose dependent. Cell progeny from the mMPC product is detected in the bone marrow, spleen, blood, and liver by flow cytometry 1 week postinfusion but is no longer evident in most animals 4 weeks posttransplant. In this model, the ex vivo-generated pooled allogeneic mMPC product (i) expands and differentiates in vivo; (ii) is functional and prevents death from invasive fungal infection; and (iii) does not permanently engraft or cause allosensitization. These data suggest that an analogous ex vivo-expanded human myeloid progenitor cell product may be an effective off-the-shelf bridging therapy for the infectious complications that develop during hematopoietic recovery following hematopoietic cell transplantation or intensive chemotherapy.

ROBO4-mediated vascular integrity regulates the directionality of hematopoietic stem cell trafficking.

Despite the use of hematopoietic stem cells (HSCs) in clinical therapy for over half a century, the mechanisms that regulate HSC trafficking, engraftment, and life-long persistence after transplantation are unclear. Here, we show that the vascular endothelium regulates HSC trafficking into and out of bone marrow (BM) niches. Surprisingly, we found that instead of acting as barriers to cellular entry, vascular endothelial cells, via the guidance molecule ROBO4, actively promote HSC translocation across vessel walls into the BM space. In contrast, we found that the vasculature inhibits the reverse process, as induced vascular permeability led to a rapid increase in HSCs in the blood stream. Thus, the vascular endothelium reinforces HSC localization to BM niches both by promoting HSC extravasation from blood-to-BM and by forming vascular barriers that prevent BM-to-blood escape. Our results uncouple the mechanisms that regulate the directionality of HSC trafficking and show that the vasculature can be targeted to improve hematopoietic transplantation therapies.

Dynamic expression of the Robo ligand Slit2 in bone marrow cell populations.

The bone marrow (BM) niche is essential for lifelong hematopoietic stem cell (HSC) maintenance, proliferation and differentiation. Several BM cell types, including osteoblast lineage cells (OBC), mesenchymal stem cells (MSC) and endothelial cells (EC) have been implicated in supporting HSC location and function, but the relative importance of these cell types and their secreted ligands remain controversial. We recently found that the cell surface receptors Robo4 and CXCR4 cooperate to localize HSC to BM niches. We hypothesized that Slit2, a putative ligand for Robo4, cooperates with the CXCR4 ligand SDF1 to direct HSC to specific BM niche sites. Here, we have isolated OBC, MSC and EC by flow cytometry and determined their frequency within the bone marrow and the relative mRNA levels of Slit2, SDF1 and Robo4. We found that expression of Slit2 and SDF1 were dynamically regulated in MSC and OBC-like populations following radiation, while Robo4 expression was restricted to EC. Radiation also significantly affected the cellularity and frequency of both the non-adherent and adherent cells within the BM stroma. These data support a physiological role for Slit2 in regulating the dynamic function of Robo-expressing cells within BM niches at steady state and following radiation.

All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells.

While it is clear that a single hematopoietic stem cell (HSC) is capable of giving rise to all other hematopoietic cell types, the differentiation paths beyond HSC remain controversial. Contradictory reports on the lineage potential of progenitor populations have questioned their physiological contribution of progenitor populations to multilineage differentiation. Here, we established a lineage tracing mouse model that enabled direct assessment of differentiation pathways in vivo. We provide definitive evidence that differentiation into all hematopoietic lineages, including megakaryocyte/erythroid cell types, involves Flk2-expressing non-self-renewing progenitors. A Flk2+ stage was used during steady-state hematopoiesis, after irradiation-induced stress and upon HSC transplantation. In contrast, HSC origin and maintenance do not include a Flk2+ stage. These data demonstrate that HSC specification and maintenance are Flk2 independent, and that hematopoietic lineage separation occurs downstream of Flk2 upregulation.

Robo4 cooperates with CXCR4 to specify hematopoietic stem cell localization to bone marrow niches.

Specific bone marrow (BM) niches are critical for hematopoietic stem cell (HSC) function during both normal hematopoiesis and in stem cell transplantation therapy. We demonstrate that the guidance molecule Robo4 functions to specifically anchor HSCs to BM niches. Robo4-deficient HSCs displayed poor localization to BM niches and drastically reduced long-term reconstitution capability while retaining multilineage potential. Cxcr4, a critical regulator of HSC location, is upregulated in Robo4(-/-) HSCs to compensate for Robo4 loss. Robo4 deletion led to altered HSC mobilization efficiency, revealing that inhibition of both Cxcr4- and Robo4-mediated niche interactions are necessary for efficient HSC mobilization. Surprisingly, we found that WT HSCs express very low levels of Cxcr4 and respond poorly to Cxcr4 manipulation relative to other hematopoietic cells. We conclude that Robo4 cooperates with Cxcr4 to endow HSCs with competitive access to limited stem cell niches, and we propose Robo4 as a therapeutic target in HSC transplantation therapy.

Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy.

BACKGROUND: Patients generally die of cancer after the failure of current therapies to eliminate residual disease. A subpopulation of tumor cells, termed cancer stem cells (CSC), appears uniquely able to fuel the growth of phenotypically and histologically diverse tumors. It has been proposed, therefore, that failure to effectively treat cancer may in part be due to preferential resistance of these CSC to chemotherapeutic agents. The subpopulation of human colorectal tumor cells with an ESA(+)CD44(+) phenotype are uniquely responsible for tumorigenesis and have the capacity to generate heterogeneous tumors in a xenograft setting (i.e. CoCSC). We hypothesized that if non-tumorigenic cells are more susceptible to chemotherapeutic agents, then residual tumors might be expected to contain a higher frequency of CoCSC. METHODS AND FINDINGS: Xenogeneic tumors initiated with CoCSC were allowed to reach approximately 400 mm(3), at which point mice were randomized and chemotherapeutic regimens involving cyclophosphamide or Irinotecan were initiated. Data from individual tumor phenotypic analysis and serial transplants performed in limiting dilution show that residual tumors are enriched for cells with the CoCSC phenotype and have increased tumorigenic cell frequency. Moreover, the inherent ability of residual CoCSC to generate tumors appears preserved. Aldehyde dehydrogenase 1 gene expression and enzymatic activity are elevated in CoCSC and using an in vitro culture system that maintains CoCSC as demonstrated by serial transplants and lentiviral marking of single cell-derived clones, we further show that ALDH1 enzymatic activity is a major mediator of resistance to cyclophosphamide: a classical chemotherapeutic agent. CONCLUSIONS: CoCSC are enriched in colon tumors following chemotherapy and remain capable of rapidly regenerating tumors from which they originated. By focusing on the biology of CoCSC, major resistance mechanisms to specific chemotherapeutic agents can be attributed to specific genes, thereby suggesting avenues for improving cancer therapy.

Reversal of autoimmune disease in lupus-prone New Zealand black/New Zealand white mice by nonmyeloablative transplantation of purified allogeneic hematopoietic stem cells.

Patients with severe systemic lupus erythematosus (SLE) refractory to conventional treatment are candidates for autologous hematopoietic stem cell (HSC) transplantation if the intent is to reset the immunologic clock. These patients might be candidates for allotransplantation with (SLE)-resistant major histocompatibility complex (MHC) haplotype-matched HSC if partial or complete replacement of an autoimmune-prone system is the intent. Using lupus-prone New Zealand black x New Zealand white (NZBW) mice, we investigated the use of highly enriched, haplomismatched, allogeneic HSC to prevent development of or to treat established autoimmune pathology. Young NZBW mice receiving purified allogeneic HSC transplants had improved survival, decreased proteinuria, circulating immune complexes, and autoantibodies to nuclear antigens than did untreated mice or mice given NZBW HSCs. NZBW mice with established lupus-like disease that received nonmyeloablative conditioning and transplants of (MHC) haplomismatched allogeneic HSCs also had greatly increased overall survival. Mice that received transplants exhibited stabilization or reversal of their lupus symptoms; stabilized or decreased proteinuria, and a lower frequency of elevated circulating immune complexes or autoantibodies than did control mice. Induction of durable mixed chimerism by transplantation of purified allogeneic HSCs after nonmyeloablative conditioning has the potential to reverse symptoms of established NZBW lupus.