Expansion Of Hematopoietic Progenitors Research Articles

Loss of Dnajc21 leads to cytopenia and altered nucleotide metabolism in zebrafish.

Mutations in the DNAJC21 gene were recently described in Shwachman-Diamond syndrome (SDS), a bone marrow failure syndrome with high predisposition for myeloid malignancies. To study the underlying biology in hematopoiesis regulation and disease, we generated the first in vivo model of Dnajc21 deficiency using the zebrafish. Zebrafish dnajc21 mutants phenocopy key SDS patient phenotypes such as cytopenia, reduced growth, and defective protein synthesis. We show that cytopenia results from impaired hematopoietic differentiation, accumulation of DNA damage, and reduced cell proliferation. The introduction of a biallelic tp53 mutation in the dnajc21 mutants leads to the development of myelodysplastic neoplasia-like features defined by abnormal erythroid morphology and expansion of hematopoietic progenitors. Using transcriptomic and metabolomic analyses, we uncover a novel role for Dnajc21 in nucleotide metabolism. Exogenous nucleoside supplementation restores neutrophil counts, revealing an association between nucleotide imbalance and neutrophil differentiation, suggesting a novel mechanism in dnajc21-mutant SDS biology.

Wiskott Aldrich syndrome protein (WASp)-deficient Th1 cells promote R-loop-driven transcriptional insufficiency and transcription-coupled nucleotide excision repair factor (TC-NER)-driven genome-instability in the pathogenesis of T cell acute lymphoblastic leukemia

BackgroundT-ALL is an aggressive hematological tumor that develops as the result of a multi-step oncogenic process which causes expansion of hematopoietic progenitors that are primed for T cell development to undergo malignant transformation and growth. Even though first-line therapy has a significant response rate, 40% of adult patients and 20% of pediatric patients will relapse. Therefore, there is an unmet need for treatment for relapsed/refractory T-ALL to develop potential targeted therapies. MethodsPediatric T-ALL patient derived T cells were grown under either nonskewingTh0 or Th1-skewing conditions to further process for ChIP-qPCR, RDIP-qPCR and other RT-PCR assays. Endogenous WASp was knocked out using CRISPR-Cas9 and was confirmed using flow cytometry and western blotting. LC-MS/MS was performed to find out proteomic dataset of WASp-interactors generated from Th1-skewed, human primary Th-cells. DNA-damage was assessed by immunofluorescence confocal-imaging and single-cell gel electrophoresis (comet assay). Overexpression of RNaseH1 was also done to restore normal Th1-transcription in WASp-deficient Th1-skewed cells. ResultsWe discovered that nuclear-WASp is required for suppressing R-loop production (RNA/DNA-hybrids) at Th1-network genes by ribonucleaseH2 (RNH2) and topoisomerase1. Nuclear-WASp is associated with the factors involved in preventing and dissolving R-loops in Th1 cells. In nuclear- WASp-reduced malignant Th1-cells, R-loops accumulate in vivo and are processed into DNA-breaks by transcription-coupled-nucleotide-excision repair (TC-NER). Several epigenetic modifications were also found to be involved at Th1 gene locus which are responsible for active/repressive marks of particular genes. By demonstrating WASp as a physiologic regulator of programmed versus unprogrammed R-loops, we suggest that the transcriptional role of WASp in vivo extends also to prevent transcription-linked DNA damage during malignancy and through modification of epigenetic dysregulations. ConclusionOur findings present a provocative possibility of resetting R-loops as a therapeutic intervention to correct both immune deficiency and malignancy in T-cell acute lymphoblastic leukemia patients and a novel role of WASp in the epigenetic regulation of T helper cell differentiation in T-ALL patients, anticipating WASp's requirement for the suppression of T-ALL progression.

Neutrophil Granulopoiesis Optimized Through Ex Vivo Expansion of Hematopoietic Progenitors in Engineered 3D Gelatin Methacrylate Hydrogels.

Neutrophils are the first line of defense of the innate immune system. In response to methicillin-resistant Staphylococcus aureus infection in the skin, hematopoietic stem, and progenitor cells (HSPCs) traffic to wounds and undergo extramedullary granulopoiesis, producing neutrophils necessary to resolve the infection. This prompted the engineering of a gelatin methacrylate (GelMA) hydrogel that encapsulates HSPCs within a matrix amenable to subcutaneous delivery. The authors study the influence of hydrogel mechanical properties to produce an artificial niche for granulocyte-monocyte progenitors (GMPs) to efficiently expand into functional neutrophils that can populate infected tissue. Lin-cKIT+ HSPCs, harvested from fluorescent neutrophil reporter mice, are encapsulated in GelMA hydrogels of varying polymer concentration and UV-crosslinked to produce HSPC-laden gels of specific stiffness and mesh sizes. Softer 5% GelMA gels yield the most viable progenitors and effective cell-matrix interactions. Compared to suspension culture, 5% GelMA results in a twofold expansion of mature neutrophils that retain antimicrobial functions including degranulation, phagocytosis, and ROS production. When implanted dermally in C57BL/6J mice, luciferase-expressing neutrophils expanded in GelMA hydrogels are visualized at the site of implantation for over 5 days. They demonstrate the potential of GelMA hydrogels for delivering HSPCs directly to the site of skin infection to promote local granulopoiesis.

Mesenchymal Stromal Cells from Perinatal Tissues as an Alternative for Ex Vivo Expansion of Hematopoietic Progenitor and Stem Cells from Umbilical Cord Blood.

Umbilical cord blood (UCB) serves as a source of hematopoietic stem and progenitor cells (HSPCs) utilized in the regeneration of hematopoietic and immune systems, forming a crucial part of the treatment for various benign and malignant hematological diseases. UCB has been utilized as an alternative HSPC source to bone marrow (BM). Although the use of UCB has extended transplantation access to many individuals, it still encounters significant challenges in selecting a histocompatible UCB unit with an adequate cell dose for a substantial proportion of adults with malignant hematological diseases. Consequently, recent research has focused on developing ex vivo expansion strategies for UCB HSPCs. Our results demonstrate that co-cultures with the investigated mesenchymal stromal cells (MSCs) enable a 10- to 15-fold increase in the cellular dose of UCB HSPCs while partially regulating the proliferation capacity when compared to HSPCs expanded with early acting cytokines. Furthermore, the secretory profile of UCB-derived MSCs closely resembles that of BM-derived MSCs. Moreover, both co-cultures exhibit alterations in cytokine secretion, which could potentially impact HSPC proliferation during the expansion process. This study underscores the fact that UCB-derived MSCs possess a remarkably similar supportive capacity to BM-derived MSCs, implying their potential use as feeder layers in the ex vivo expansion process of HSPCs.

Complete miRNA-15/16 loss in mice promotes hematopoietic progenitor expansion and a myeloid-biased hyperproliferative state

Dysregulated apoptosis and proliferation are fundamental properties of cancer, and microRNAs (miRNA) are critical regulators of these processes. Loss of miR-15a/16-1 at chromosome 13q14 is the most common genomic aberration in chronic lymphocytic leukemia (CLL). Correspondingly, the deletion of either murine miR-15a/16-1 or miR-15b/16-2 locus in mice is linked to B cell lymphoproliferative malignancies. However, unexpectedly, when both miR-15/16 clusters are eliminated, most double knockout (DKO) mice develop acute myeloid leukemia (AML). Moreover, in patients with CLL, significantly reduced expression of miR-15a, miR-15b, and miR-16 associates with progression of myelodysplastic syndrome to AML, as well as blast crisis in chronic myeloid leukemia. Thus, the miR-15/16 clusters have a biological relevance for myeloid neoplasms. Here, we demonstrate that the myeloproliferative phenotype in DKO mice correlates with an increase of hematopoietic stem and progenitor cells (HSPC) early in life. Using single-cell transcriptomic analyses, we presented the molecular underpinning of increased myeloid output in the HSPC of DKO mice with gene signatures suggestive of dysregulated hematopoiesis, metabolic activities, and cell cycle stages. Functionally, we found that multipotent progenitors (MPP) of DKO mice have increased self-renewing capacities and give rise to significantly more progeny in the granulocytic compartment. Moreover, a unique transcriptomic signature of DKO MPP correlates with poor outcome in patients with AML. Together, these data point to a unique regulatory role for miR-15/16 during the early stages of hematopoiesis and to a potentially useful biomarker for the pathogenesis of myeloid neoplasms.

Modeling Clonal Progression in SF3B1-Mutant Myelodysplastic Syndrome

SF3B1-mutant myelodysplastic syndrome (MDS) has recently been proposed as a distinct disorder characterized by ring sideroblasts, ineffective erythropoiesis and good prognosis. Selected co-occurring genetic abnormalities were reported associated with significantly worse outcome and suggested as exclusion criteria for the proposed entity. However, it remains unclear how a limited spectrum of co-occurring drivers affects SF3B1-mutant MDS biology to determine evolution from a relatively indolent condition to high risk malignancy.To gain a better insight into the clonal progression of SF3B1-mutant MDS, we analyzed SF3B1 co-mutations in a cohort of 176 SF3B1-mutated patients diagnosed with a myeloid neoplasm. RUNX1 and STAG2 were the only co-mutated genes found significantly associated with advanced disease phenotype (i.e. MDS with excess blasts and secondary acute myeloid leukemia) (OR=18.36 (2.18-862.91), P=0.001, and OR= Inf (3.57-Inf), P<.001, respectively), and reduced overall survival (P<.001). Based on these data, we hypothesized that acquisition of RUNX1 or STAG2 co-mutations in patients with SF3B1-mutant MDS drives progression to high-risk neoplasms.RUNX1 and STAG2 control hematopoietic stem and progenitor cell self-renewal and differentiation by regulation of gene expression. To explore the biological impact of RUNX1 or STAG2 loss in the context of SF3B1-mutant MDS, we disrupted RUNX1 or STAG2 using CRISPR/Cas9 in an induced pluripotent stem cell (iPSC) model of MDS-RS that we previously established by reprogramming of bone marrow cells from SF3B1-mutant individuals. This patient-derived system allows hematopoietic progenitor expansion through doxycycline-mediated expression of 5 transcription factors (5F-HPC) and multilineage differentiation upon doxycycline withdrawal. We asked how RUNX1 or STAG2 disruption affected SF3B1-mutant HPCs self-renewal and differentiation in our model of MDS-RS, and sought to define the underlying transcriptional changes.RUNX1-edited cells maintained significantly higher proportion of CD34 + HPCs, suggesting that RUNX1 mutation increases self-renewal capacity of SF3B1-mutant progenitors. Consistently, SF3B1/RUNX1 double-mutant 5F-HPCs showed positive enrichment of HSC-specific gene signatures. This was accompanied by broad upregulation of inflammatory programs, recapitulating the activated gene signatures we identified in SF3B1/RUNX1 co-mutated patients in our cohort. RUNX1 disruption promoted myeloid skewing at the expense of erythroid differentiation in SF3B1-mutant cells. Consistent with this, granulocyte-monocyte progenitors (GMP) and myelo-erythroid transcriptional programs were positively and negatively enriched, respectively. By contrast, in SF3B1/STAG2 double-mutant 5F-HPCs, both erythroid and myeloid populations were reduced compared to SF3B1 single mutant controls, suggesting a block in both myeloid and erythroid differentiation, despite the presence of pro-differentiation signals. This was supported by a profound down-regulation of genes involved in the response to external stimulus and suppression of GMP specific transcriptional signature.In summary, we identified RUNX1 and STAG2 mutations as main drivers of disease progression in SF3B1-mutant patients, and generated extensive cell line panels to interrogate their functional interaction with mutant SF3B1. By applying CRISPR/Cas9 editing to our MDS-RS model, we could overcome limited availability of primary MDS samples to show that RUNX1 or STAG2 co-mutations drive progression through distinct biological mechanisms in SF3B1-mutant HPCs. DisclosuresNo relevant conflicts of interest to declare.

Identification and Molecular Characterization of Endothelial Cells Associated with Hematopoietic Niche Formation in Humans Identifies IL-33 As an Anabolic Niche Factor

Mammalian bone marrow (BM) regeneration after myeloablation requires orchestrated interplay between osteogenesis, angiogenesis and hematopoiesis. Specific subsets of endothelial cells (ECs) have been identified in mice, including sinusoidal ECs (Hooper et al., 2009) and H-endothelium (Kusumbe et al., 2014, 2016; Ramasamy et al., 2014), that drive and coordinate these processes. Their relevance to human biology, however, remains largely elusive.Here, using a combinatory approach of cell purification and transcriptomics, we identify and characterize EC subsets associated with BM niche formation during ontogeny and regeneration in humans.Massive parallel RNA sequencing of FACS-purified BM ECs (CD45-CD235-7AAD-CD31+CD9+) from AML patients in the regenerating phase after chemotherapy (n=3) revealed upregulated endoglin (CD105) expression, in comparison to ECs at diagnosis or from normal BM (n=7) (FPKM fold-change increase (FCI): 10.1 ± 4.9; GLM LRT Edge R; p=0.001). FACS analysis confirmed a strong enrichment of endoglin-expressing ECs in regenerative marrow (n=48) compared to normal BM (n=9) (10.81% ± 2.14 vs 0.48% ± 0.24 of CD31+CD9+ cells; p=0.043).Similarly, in human fetal BM at 15-20 weeks after gestation (n=15), when angiogenesis, osteogenesis and hematopoiesis are tightly coupled, a striking predominance of endoglin-expressing ECs (62.8% ± 5.9 of CD31+CD9+ cells) was found, confirmed by in situ immunohistochemistry in comparison to adult core hip bone biopsies. Comprehensive transcriptional characterization by RNAseq of these endoglin-expressing ‘human regeneration-associated EC’ (hREC) from fetal bone (n=7) in comparison to endoglin- ECs revealed a remarkable phenotypic and molecular similarity to murine H-endothelium as well as activation of pathways and factors implicated in hematopoiesis, osteogenesis and angiogenesis. These included upregulated expression of CD31 (PECAM1) (FPKM FCI: 4.7 ± 1.0; p=0.04), mucin-like sialoglycoprotein endomucin (EMCN) (FPKM FCI: 435 ± 112; p <0.001), stromal cell-derived factor 1 (CXCL12) (FPKM FCI: 108 ± 34; p <0.001) and transcriptional activation of NOTCH (Normalized Enrichment score (NES): 1.51, False Discovery Rate-adjusted q-value (FDR): <0.10) and HIF1a (NES: 1.60, FDR <0.10) signaling pathways.IL-33 was found to be highly and significantly overexpressed in both fetal and regenerative marrow ECs in comparison to steady-state normal BM ECs (FPKM FCI: 587 ± 128 and 174 ± 86, respectively, both p <0.001). Expression of IL-33 in endoglin-expressing ECs was confirmed at the protein level in both humans and mice. Recombinant human IL-33 (1) promoted the expansion of distinct subsets of hematopoietic precursor cells, specifically immunophenotypic multipotent progenitors (2.5-FCI ± 0.43; p<0.05) and multilymphoid progenitors (2.7-FCI ± 0.2; p<0.01), (2) increased metabolic activity in HUVECs (PrestoBlue Cell Viability assay; mean fluorescence intensity 1.40-FCI ± 0.02; p=0.03), and (3) promoted osteogenic differentiation of BM-derived mesenchymal cells (Alizarin red-staining and colorimetric assessment of calcium deposition (3.61-FCI ± 0.63; p<0.02)). Repressing IL-33 in HUVEC by shRNAs impaired their expansion in culture dramatically (FC decrease 6-7 fold; p=0.01). In vivo, enrichment of an endoglin-expressing subset of BM ECs was observed in mice exposed to a myeloablative dose of 5-fluorouracil (n=5) compared to steady-state mice (n=3) (FCI: 9.0 ± 1.7; p=0.01). Administration of recombinant murine IL-33 to C57BL/6 wild-type mice recapitulated the expansion of hematopoietic progenitors with myeloid and lymphoid potential (HPC1 subset) (FCI 2.6 ± 0.5; p=0.003), and resulted in a relative increase in CD31+CD105+ BM ECs (FCI 3.5 ± 0.3; p=0.002) as well as bone progenitor (Lin-Ter119-CD51+Sca-) cells (FCI 2.9 ± 0.7; p=0.06) within the niche.Collectively, the data reveal the existence of an endothelial subset associated with BM regeneration and ontogeny in humans and identify IL-33 as a potential driver of marrow recovery. The molecular characterization of hREC is, thus, anticipated to instruct the discovery of novel determinants of EC-driven regeneration of the hematopoietic system after injury. DisclosuresRaaijmakers:Novartis: Consultancy.

TET2 Driven Clonal Hematopoiesis Promotes Allograft Tolerance in a Mouse Heart Transplant Model

Purpose Clonal hematopoiesis (CH) is the result of clonal expansion of hematopoietic progenitors within the bone marrow (BM) that harbor mutations that confer fitness advantage relative to the background population. These clones can expand to become a dominant source of peripheral blood cells. Clonal mutations in TET2 (chromatin demethylase) have the strongest association with cardiovascular mortality and are prevalent in heart failure patients. Increased production of cytokines (i.e., interleukin-1β) by monocytes and macrophages may represent the underlying mechanism by which TET2 CH contributes to accelerated atherosclerosis and heart failure progression. The impact of TET2 CH on heart transplantation remains unexplored. Methods We established an adoptive BM transplantation CH model. Unfractionated BM (1.5 × 107cells) harvested from C57/B6 control or TET2-/- mice was injected into syngeneic C57/B6 UBC-GFP animals on three consecutive days. The extent of peripheral chimerism was assessed by flow cytometry 4-8 weeks after adoptive transfer. We then transplanted Balb/c donor hearts into chimeric C57/B6 recipient mice. All mice received CTLA4 Ig (days 0, 2 4, 6) for immunosuppression. The primary endpoint was a time to event analysis of allograft survival assessed by daily palpation. Secondary endpoints included histology, immunohistochemistry, and measurement of donor alloreactive T-cells and antibodies. Results Adoptive transfer of C57/B6 control BM into C57/B6 UBC-GFP mice led to diminished chimerism (0.6% - 3.6%) compared to adoptive transfer of C57/B6 TET2-/- BM into C57/B6 UBC-GFP mice (32 - 34.6%). We observed marked differences in donor heart survival between experimental groups. Following transplantation, recipient mice that received control BM displayed an average survival of 18 days, whereas recipient mice that received TET2-/- BM demonstrated evidence of long-term survival with 2/3 of mice surviving beyond 60 days (p Conclusion TET2 driven CH confers protection from allograft rejection and promotes long-term allograft acceptance in mice.

Bone Marrow Homeostasis Is Impaired via JAK/STAT and Glucocorticoid Signaling in Cancer Cachexia Model.

Simple SummaryCancer cachexia is a systemic inflammatory disease characterized by the loss of muscle and fat and occurs in 50–80% of cancer patients. In cancer cachexia, the tumor tissues interact with other tissues and organs using secretory factors. Differentiated immune cells from hematopoietic stem cells (HSCs) of the bone marrow contribute to the systemic inflammation and may be affected by these intertissue interactions. However, the significant changes that occur in the bone marrow and the underlying mechanisms are still unclear. Here, we investigated the effects of cancer cachexia on bone and stem cells that reside in the bone marrow using a lung cancer cachexia animal model. Cancer cachexia induces bone loss and impairs the properties of the bone marrow mesenchymal stem cells via JAK/STAT and glucocorticoid signaling. Our findings provide new insights for developing a novel therapeutic strategy for cancer cachexia.Cancer cachexia is a multifactorial systemic inflammation disease caused by complex interactions between the tumor and host tissues via soluble factors. However, whether cancer cachexia affects the bone marrow, in particular the hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), remains unclear. Here, we investigated the bone marrow and bone in a cancer cachexia animal model generated by transplanting Lewis lung carcinoma cells. The number of bone marrow mononuclear cells (BM-MNCs) started to significantly decrease in the cancer cachectic animal model prior to the discernable loss of muscle and fat. This decrease in BM-MNCs was associated with myeloid skewing in the circulation and the expansion of hematopoietic progenitors in the bone marrow. Bone loss occurred in the cancer cachexia animal model and accompanied the decrease in the bone marrow MSCs that play important roles in both supporting HSCs and maintaining bone homeostasis. Glucocorticoid signaling mediated the decrease in bone marrow MSCs in the cancer cachectic environment. The cancer cachexia environment also skewed the differentiation of the bone marrow MSCs toward adipogenic fate via JAK/STAT as well as glucocorticoid signaling. Our results suggest that the bone loss induced in cancer cachexia is associated with the depletion and the impaired differentiation capacity of the bone marrow MSCs.

TLR7/8 agonist treatment induces an increase in bone marrow resident dendritic cells and hematopoietic progenitor expansion and mobilization

There is accumulating evidence suggesting that toll-like receptor (TLR) signals play an important role in the regulation of hematopoietic stem/progenitor cells (HSPCs). TLR7/8 stimulation induces the myeloid differentiation of normal HSPCs and acute myeloid leukemia cells. However, the in vivo effect of TLR7/8 agonists on hematopoiesis is largely unknown. Here, we show that, similar to TLR4 and TLR2, treatment with the TLR7/8 agonist R848 induces an expansion of phenotypic hematopoietic stem cells (HSCs) with reduced repopulating potential and HSPC mobilization. In contrast to chronic TLR4 stimulation, treatment with R848 for 5 days did not induce a significant increase in myeloid-biased HSCs. Treatment with R848 results in a significant increase in classic dendritic cells (DCs) in the bone marrow, but a decrease in common dendritic cell progenitors and pre-DCs. Phenotypic analysis of DCs revealed that R848 treatment is associated with altered expression of certain chemokines, activation markers, and migratory receptors. Together, these data indicate that systemic administration of a TLR7/8 agonist has unique effects on hematopoiesis, including the expansion of DCs in the bone marrow, that might have clinical relevance to augment responses to certain immunotherapies, such as cancer vaccines and immune checkpoint blockade.

Bone marrow CX3CR1+ mononuclear cells relay a systemic microbiota signal to control hematopoietic progenitors in mice

AbstractThe microbiota regulate hematopoiesis in the bone marrow (BM); however, the detailed mechanisms remain largely unknown. In this study, we explored how microbiota-derived molecules (MDMs) were transferred to the BM and sensed by the local immune cells to control hematopoiesis under steady-state conditions. We reveal that MDMs, including bacterial DNA (bDNA), reach the BM via systemic blood circulation and are captured by CX3CR1+ mononuclear cells (MNCs). CX3CR1+ MNCs sense MDMs via endolysosomal Toll-like receptors (TLRs) to produce inflammatory cytokines, which control the basal expansion of hematopoietic progenitors, but not hematopoietic stem cells, and their differentiation potential toward myeloid lineages. CX3CR1+ MNCs colocate with hematopoietic progenitors at the perivascular region, and the depletion of CX3CR1+ MNCs impedes bDNA influx into the BM. Moreover, the abrogation of TLR pathways in CX3CR1+ MNCs abolished the microbiota effect on hematopoiesis. These studies demonstrate that systemic MDMs control BM hematopoiesis by producing CX3CR1+ MNC–mediated cytokines in the steady-state.

Trauma Induces Emergency Hematopoiesis through IL-1/MyD88-Dependent Production of G-CSF.

The inflammatory response to infection or injury dramatically increases the hematopoietic demand on the bone marrow to replace effector leukocytes consumed in the inflammatory response. In the setting of infection, pathogen-associated molecular patterns induce emergency hematopoiesis, activating hematopoietic stem and progenitor cells to proliferate and produce progeny for accelerated myelopoiesis. Sterile tissue injury due to trauma also increases leukocyte demand; however, the effect of sterile tissue injury on hematopoiesis is not well described. We find that tissue injury alone induces emergency hematopoiesis in mice subjected to polytrauma. This process is driven by IL-1/MyD88-dependent production of G-CSF. G-CSF induces the expansion of hematopoietic progenitors, including hematopoietic stem cells and multipotent progenitors, and increases the frequency of myeloid-skewed progenitors. To our knowledge, these data provide the first comprehensive description of injury-induced emergency hematopoiesis and identify an IL-1/MyD88/G-CSF-dependent pathway as the key regulator of emergency hematopoiesis after injury.

Trauma Induces Emergency Hematopoiesis through IL-1/MyD88-Dependent Production of G-CSF

Abstract The acute inflammatory response to infection or injury dramatically increases the hematopoietic demand on the bone marrow to replace effector leukocytes consumed in the inflammatory response. In the setting of infection, pathogen-associated molecular patterns induce emergency hematopoiesis, activating hematopoietic stem and progenitor cells to proliferate, thereby providing progeny for accelerated myelopoiesis. Sterile tissue injury due to trauma also increases leukocyte demand; however, the effect of sterile tissue injury on hematopoiesis is not well described. We used a mouse model of multisystem injury to investigate the effects of these injuries on bone marrow progenitor frequencies and phenotypes. We find that tissue injury alone induces emergency hematopoiesis in mice subjected to polytrauma. This process is driven by IL-1/MyD88-dependent production of G-CSF. G-CSF induces expansion of hematopoietic progenitors including hematopoietic stem cells and multipotent progenitors and increases the frequency of myeloid-skewed progenitors. These data provide the first comprehensive description of injury-induced emergency hematopoiesis and identify an IL-1/MyD88/G-CSF dependent pathway as the key regulator of emergency hematopoiesis after injury.

Effects of Delta-Like Noncanonical Notch Ligand 1 Expression of Human Fetal Liver Hepatoblasts on Hematopoietic Progenitors.

Although the hepatic and hematopoietic progenitors of the liver are well characterized, the interactions between these two lineages remain mostly elusive. Hepatoblasts express delta-like noncanonical Notch ligand 1 (Dlk1), whose cleaved extracellular domain can become a soluble protein. We assessed the effects of DLK1 gene expression knockdown in cultures of total fetal liver cells. Furthermore, we separated Dlk1+ hepatoblasts from the total liver cell fraction and investigated effects of direct cell contact. Dlk1− cells were cultured either without Dlk1+ hepatoblasts, in direct contact with hepatoblasts, or separated from hepatoblasts by a porous membrane in inserts to inhibit cell contact but allow free exchange of molecules. Expression of the hepatic and hematopoietic genes, colony forming unit potential of various hematopoietic progenitors, and cell numbers and types were investigated. We found that DLK1 knockdown in total fetal liver cell cultures decreased total cell numbers. The expression of hepatic progenitor genes and mature hematopoietic genes was affected. Hematopoietic BFU-E and CFU-GM colony numbers were reduced significantly. The depletion of Dlk1+ hepatoblasts in culture decreased the potential of all hematopoietic progenitors to form colonies of all types and reduced the percentage of mature hematopoietic cells. The addition of hepatoblasts in inserts to Dlk1− cells further decreased the potential to form the CFU-GM and CFU-GEMM colonies and the percentage of mature hematopoietic cells but increased total cell numbers. Conclusively, direct contact of Dlk1 supports hematopoietic progenitor expansion and functionality that cannot be reconstituted in coculture without direct cell contact.

Phthalates affect the in vitro expansion of human hematopoietic stem cell.

Phthalates are esters of phthalic acid used industrially as plastic additives, however, these are not covalently bound to the polymer matrix and therefore can be released to the environment. The aim of this study was to evaluate the effect of four phthalates: dibutyl phthalate (DBP), benzyl butyl phthalate (BBP), diethyl phthalate (DEP) and diethylhexyl phthalate (DEHP) on the in vitro expansion of human hematopoietic cells from umbilical cord blood. For this, 0.5 × 106 cells/mL were exposure to concentrations ranging from 0.1 to 100μg/mL and the total cell expansion was determined after 14days of culture in IMDM-cytokines medium. The control cultures attained 1.31 ± 0.21 × 106 cell/mL, whereas the cultures exposed to DBP, BBP and DEHP showed a reduction from 23 to 81%, 17 to 69% and 15 to 93.5%, respectively. DEP did not affect the total cell expansion. The most significant decrease on total cell expansion was observed at 0.1μg/mL DBP, 100μg/mL BBP and 10μg/mL DEHP (p < 0.05). Additionally, the effect of these compounds on the expansion of hematopoietic progenitors was analyzed by clonogenic assays as colony forming units (CFU). The CFU decreased considerably compared with respect to the control cultures. The reduction was 74.6 and 99.1% at 10 and 100μg/mL DBP respectively, whereas 100μg/mL BBP and 100μg/mL DEHP reduced the CFU expansion in 97.1% and 81%, respectively. Cultures exposed to DEP did not show significant differences. The results demonstrate the toxicity of DBP, BBP and DEHP on the human hematopoietic stem cells.

Nfe2 is dispensable for early but required for adult thrombocyte formation and function in zebrafish

AbstractThe NFE2 transcription factor is expressed in multiple hematopoietic lineages with a well-defined role in regulating megakaryocyte biogenesis and platelet production in mammals. Mice deficient in NFE2 develop severe thrombocytopenia with lethality resulting from neonatal hemorrhage. Recent data in mammals reveal potential differences in embryonic and adult thrombopoiesis. Multiple studies in zebrafish have revealed mechanistic insights into hematopoiesis, although thrombopoiesis has been less studied. Rather than platelets, zebrafish possess thrombocytes, which are nucleated cells with similar functional properties. Using transcription activator-like effector nucleases to generate mutations in nfe2, we show that unlike mammals, zebrafish survive to adulthood in the absence of Nfe2. Despite developing severe thrombocytopenia, homozygous mutants do not display overt hemorrhage or reduced survival. Surprisingly, quantification of circulating thrombocytes in mutant 6-day-old larvae revealed no significant differences from wild-type siblings. Both wild-type and nfe2 null larvae formed thrombocyte-rich clots in response to endothelial injury. In addition, ex vivo thrombocytic colony formation was intact in nfe2 mutants, and adult kidney marrow displayed expansion of hematopoietic progenitors. These data suggest that loss of Nfe2 results in a late block in adult thrombopoiesis, with secondary expansion of precursors: features consistent with mammals. Overall, our data suggest parallels with erythropoiesis, including distinct primitive and definitive pathways of development and potential for a previously unknown Nfe2-independent pathway of embryonic thrombopoiesis. Long-term homozygous mutant survival will facilitate in-depth study of Nfe2 deficiency in vivo, and further investigation could lead to alternative methodologies for the enhancement of platelet production.

Hematopoiesis-supportive function of growth-arrested human adipose-tissue stromal cells under physiological hypoxia

Exvivo expansion of hematopoietic progenitors is considered as an attractive tool to increase the number of stem and progenitor cells (HSPCs) for cell therapy. The efficacy of exvivo expansion is strongly depends on the feeder cell activity to mimic hematopoietic microenvironment. Here we demonstrated, that combination of mitomycin C-induced growth arrest and tissue-related O2 (physiological hypoxia) modulated stromal capacity of adipose tissue derived stromal cells (ASCs). Growth arrest did not affect viability, stromal phenotype and multilineage potential of ASCs permanently expanded at tissue-related O2. Meanwhile, the PCR analysis revealed an up-regulation of genes, encoded molecules of cell-cell (ICAM1, HCAM/CD44) and cell-matrix adhesion (ITGs), extracellular matrix production (COLs) and remodeling (MMPs, HAS1) in growth-arrested ASCs at physiological hypoxia in comparison with ambient O2 (20%). The number of ICAM-1 positive ASCs was increased under low O2 as well. These alterations contributed into the exvivo expansion of cord blood HSPCs providing the preferential production of primitive HSPCs. The number of cobblestone area forming cell (CAFC) colonies was 1.5-fold higher at physiological hypoxia (p<0.05). CAFCs considered as long-term culture-initiating cells (LTC-IC) known to support long-term hematopoiesis restoration invivo. The presented data may be applicable in the development of upscale protocols of HSPC expansion.

Immunomodulatory drugs downregulate IKZF1 leading to expansion of hematopoietic progenitors with concomitant block of megakaryocytic maturation.

The immunomodulatory drugs, lenalidomide and pomalidomide yield high response rates in multiple myeloma patients, but are associated with a high rate of thrombocytopenia and increased risk of secondary hematologic malignancies. Here, we demonstrate that the immunomodulatory drugs induce self-renewal of hematopoietic progenitors and upregulate megakaryocytic colonies by inhibiting apoptosis and increasing proliferation of early megakaryocytic progenitors via down-regulation of IKZF1. In this process, the immunomodulatory drugs degrade IKZF1 and subsequently down-regulate its binding partner, GATA1. This results in the decrease of GATA1 targets such as ZFPM1 and NFE2, leading to expansion of megakaryocytic progenitors with concomitant inhibition of maturation of megakaryocytes. The down-regulation of GATA1 further decreases CCND1 and increases CDKN2A expression. Overexpression of GATA1 abrogated the effects of the immunomodulatory drugs and restored maturation of megakaryocytic progenitors. Our data not only provide the mechanism for the immunomodulatory drugs induced thrombocytopenia but also help to explain the higher risk of secondary malignancies and long-term cytopenia induced by enhanced cell cycling and subsequent exhaustion of the stem cell pool.

Hematopoietic Niche - Exploring Biomimetic Cues to Improve the Functionality of Hematopoietic Stem/Progenitor Cells.

The adult bone marrow (BM) niche is a complex entity where a homeostatic hematopoietic system is maintained through a dynamic crosstalk between different cellular and non-cellular players. Signaling mechanisms triggered by cell-cell, cell-extracellular matrix (ECM), cell-cytokine interactions, and local microenvironment parameters are involved in controlling quiescence, self-renewal, differentiation, and migration of hematopoietic stem/progenitor cells (HSPC). A promising strategy to more efficiently expand HSPC numbers and tune their properties ex vivo is to mimic the hematopoietic niche through integration of adjuvant stromal cells, soluble cues, and/or biomaterial-based approaches in HSPC culture systems. Particularly, mesenchymal stem/stromal cells (MSC), through their paracrine activity or direct contact with HSPC, are thought to be a relevant niche player, positioning HSPC-MSC co-culture as a valuable platform to support the ex vivo expansion of hematopoietic progenitors. To improve the clinical outcome of hematopoietic cell transplantation (HCT), namely when the available HSPC are present in a limited number such is the case of HSPC collected from umbilical cord blood (UCB), ex vivo expansion of HSPC is required without eliminating the long-term repopulating capacity of more primitive HSC. Here, we will focus on depicting the characteristics of co-culture systems, as well as other bioengineering approaches to improve the functionality of HSPC ex vivo.

Human Bone Marrow Mesenchymal Stromal Cell-Derived Osteoblasts Promote the Expansion of Hematopoietic Progenitors Through Beta-Catenin and Notch Signaling Pathways.

Coculture of hematopoietic stem cells (HSC) with primary stromal cells from HSC niches supports the maintenance and expansion of HSC and progenitors ex vivo. However, a major drawback is the availability of primary human samples for research and clinical applications. We investigated the use of in vitro derived osteoblasts as a new source of feeder cells and characterized the molecular pathways that mediate their growth-promoting activities. First, we compared the growth and differentiation modulating activities of mesenchymal stromal cells (MSC)-derived osteoblasts (M-OST) with those of their undifferentiated precursor on umbilical cord blood (UCB) progenitors. Feeder-free cultures were also included as baseline control. Cell growth and expansion of hematopoietic progenitors were significantly enhanced by both feeder cell types. However, progenitor cell growth was considerably greater with M-OST. Coculture also promoted the maintenance of immature CD34+ progenitor subsets and modulated in a positive fashion the expression of several homing-related cell surface receptors, in a feeder-specific fashion. Serial transplantation experiments revealed that M-OST coculture supported the maintenance of long-term lympho-myeloid reconstituting HSC that provided engraftment levels that were generally superior to those from MSC cocultures. Mechanistically, we found that coculture with M-OST was associated with enhanced beta-catenin (β-Cat) activity in UCB cells and that abrogation of β-Cat/T-cell factor activity blunted the growth-promoting activity of the M-OST coculture. Conversely, Notch inhibition reduced UCB cell expansion, but to a much lesser extent. In conclusion, this study demonstrates that M-OST are excellent feeder cells for HSC and progenitors, and it identifies key molecular pathways that are responsible for the growth-enhancing activities of osteoblasts on UCB progenitors.