scholarly journals A new murine model of Barth Syndrome neutropenia links TAFAZZIN deficiency to increased ER stress induced apoptosis

2022 ◽  
Author(s):  
Jihee Sohn ◽  
Jelena Milosevic ◽  
Thomas Brouse ◽  
Najihah Aziz ◽  
Jenna Elkhoury ◽  
...  
Keyword(s):  
Er Stress ◽  
Murine Model ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Model System ◽  
Barth Syndrome ◽  
Loss Of Function ◽  
Inner Mitochondrial Membrane ◽  
Hematopoietic Stem ◽  
Increased Sensitivity

Barth syndrome is an inherited X-linked disorder that leads to cardiomyopathy, skeletal myopathy and neutropenia. These symptoms result from the loss of function of the enzyme TAFAZZIN, a transacylase located in the inner mitochondrial membrane that is responsible for the final steps of cardiolipin production. The link between defective cardiolipin maturation and neutropenia remains unclear. To address potential mechanisms of neutropenia, we examined myeloid progenitor development within the fetal liver of TAFAZZIN knock-out animals as well as within the adult bone marrow of wild-type recipients transplanted with TAFAZZIN KO hematopoietic stem cells. We also used the ER Hoxb8 system of conditional immortalization to establish a new murine model system for the ex vivo study of TAFAZZIN-deficient neutrophils. The TAFAZZIN KO cells demonstrated the expected dramatic differences in cardiolipin maturation that result from a lack of TAFAZZIN enzyme activity. Contrary to our hypothesis, we did not identify any significant differences in neutrophil development or neutrophil function across a variety of assays including phagocytosis, and the production of cytokines or reactive oxygen species. However, transcriptomic analysis of the TAFAZZIN-deficient neutrophil progenitors demonstrated an upregulation of markers of endoplasmic reticulum stress and confirmatory testing demonstrated that the TAFAZZIN-deficient cells had increased sensitivity to certain ER stress mediated and non ER stress mediated triggers of apoptosis. While the link between increased sensitivity to apoptosis and the variably penetrant neutropenia phenotype seen in some Barth syndrome patients remains to be clarified, our studies and new model system set a foundation for further investigation.

scholarly journals What Links Neutropenia to Immature Cardiolipin in Patients with Barth Syndrome (tafazzin-deficiency)?

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3579-3579
Author(s):  
Jihee Sohn ◽  
Thomas Brouse ◽  
Najihah Aziz ◽  
David B Sykes
Keyword(s):  
Er Stress ◽  
Knockout Mouse ◽  
Mitochondrial Membrane ◽  
Fetal Liver ◽  
Liver Cells ◽  
Barth Syndrome ◽  
Developmental Defect ◽  
Wild Type ◽  
Inner Mitochondrial Membrane ◽  
Fetal Liver Cells

Barth syndrome is an inherited X-linked disorder characterized by cardiomyopathy, skeletal muscle myopathy, and neutropenia. The syndrome arises because of inherited mutations in the gene TAZ, resulting in a loss of function of the protein tafazzin. Of note, a group of investigators recently described how tafazzin can regulate 'stemness' in models of acute myeloid leukemia (Cell Stem Cell, 2019). Tafazzin is an enzyme that processes the final step of cardiolipin maturation, replacing saturated with unsaturated acyl chains. Cardiolipin is a 4-tailed phospholipid that is almost-exclusively found in the inner membrane of the mitochondria. The lack of tafazzin activity results in a cardiolipin pool that contains more highly saturated lipid tails and it is this lack of unsaturated cardiolipins that contributes to a disorganized inner mitochondrial membrane. The link between tafazzin-deficiency and myopathy is generally explained by the dependence of muscle cells on mitochondrial function as well as oxidative respiration. The components of the electron transport chain are co-localized with cardiolipin in the inner mitochondrial membrane, and it is felt that their appropriate organization within the membrane lipid bilayer is dependent on the presence of mature cardiolipin which is lacking in those individuals with Barth syndrome. The link between tafazzin-deficiency and neutropenia is less clear. Neutrophils are terminally-differentiated effector cells of the innate immune system. They are critical for protection against bacterial and fungal pathogens and patients without sufficient neutrophils are among the most immunocompromised and at risk of lethal infection. Neutrophils have few mitochondria at baseline and are generally believed to rely primarily on glycolysis for energy production. It is not known if the mechanism of neutropenia in Barth syndrome is due to a lack of production or due to increased clearance (e.g. more prone to apoptosis). We undertook the study of tafazzin-deficient neutrophils to try to elucidate the mechanism of neutropenia in patients with Barth syndrome. We took advantage of an existing tafazzin-knockout mouse and a system of conditional immortalization of granulocyte-monocyte progenitors (GMP) using the ER-Hoxb8 system pioneered in our laboratory. This ER-Hoxb8 system allows for the unlimited ex vivo expansion of myeloid progenitors in the presence of estradiol and active Hoxb8. Once estradiol is removed from culture media, the Hoxb8 protein is inactive and the cells undergo normal, synchronous and terminal neutrophilic differentiation. In this manner, we were able to generate tafazzin-wild-type and knockout GMP lines from murine fetal liver cells. Analysis of the myeloid progenitor compartment in fetal liver cells (d14.5-d16.5) showed no difference between wild-type and knockout mice, arguing against a developmental defect (E15 results shown in PANEL A). Furthermore, the tafazzin-deficient ER-Hoxb8 GMPs and neutrophils were remarkably normal when tested across a variety of assays including phagocytosis, cytokine production and ROS generation (ROS by H2DCFDA shown in PANEL B). We hypothesized that the unpredictable neutropenia in patients with Barth Syndrome might be due to an increased proclivity to apoptosis because of the mitochondrial membrane defect. Indeed, the tafazzin-deficient GMPs showed an increased sensitivity to Bcl2-inhibition following treatment with ABT199 (PANEL C). Two lines of evidence have suggested that the increased tendency towards apoptosis may be due to endoplasmic-reticulum (ER) stress. (1) Transmission electron microscopy demonstrated 'swollen' ER in the tafazzin-deficient cells (not shown) and (2) a comparison of gene expression patterns demonstrated an increased expression of ATF4 and CHOP (DDIT3) in the tafazzin-deficient cells (PANEL D). We are now focused on validating these findings and in establishing models to confirm the ER-stress phenotype in vivo in the TAZ-knockout mouse model as well as primary samples from patients with Barth Syndrome. We hope that this line of work will confirm the mechanism of neutropenia and shed light on potential targets for therapeutic intervention. In addition, this very rare disorder has provided insight into a previously-unexpected link between neutrophil survival and the membrane integrity of the inner mitochondrial membrane. Figure Disclosures Sykes: Clear Creek Bio: Equity Ownership, Other: Co-Founder.

Taurine-Conjugated Bile Acids Protect Expanding Hematopoietic Stem/Progenitor Cells from Unfolded Protein Stress As Natural Chaperones

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4318-4318 ◽  
Author(s):  
Valgardur Sigurdsson ◽  
Hajime Takei ◽  
Svetlana Soboleva ◽  
Takashi Iida ◽  
Hiroshi Nittono ◽  
...  
Keyword(s):  
Bile Acid ◽  
Er Stress ◽  
Bile Acids ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Adult Liver ◽  
Hematopoietic Stem ◽  
Pregnant Mice

Abstract Hematopoietic stem cells (HSCs) give rise to all lineages of hematopoietic cells in the body for entire life span and are thus protected from risk factors by multiple defense systems. We have recently discovered that HSCs are highly susceptible to stress caused by accumulation of mis-/un-folded proteins, so called endoplasmic reticulum (ER) stress upon enhanced growth conditions, and addition of a specific type of bile acid (BA), Tauroursodeoxycholic acid (TUDCA), known as a chemical chaperone can maintain functional murine HSCs for 2 weeks in vitro, by reducing ER stress (Miharada et al., Cell Rep. 2014). This work depicts the importance of proper protein quality control in HSC maintenance, particularly during the expansion. HSCs are kept in dormant state in the adult body, but actively expanding in the fetal liver. BAs are synthesized from cholesterol in the liver. Interestingly, bile acid synthesis is highly up-regulated in the fetal liver during embryogenesis and the composition of fetal BAs gradually reduces after birth. In addition, composition of bile acids in the fetus is different from adult liver, with the vast majority of fetal BAs are of Taurine-conjugated form that is more stable and non-toxic. Of note, hematopoietic cells and hepatocytes producing BAs are in close contact in the fetal liver and HSCs are therefore exposed to BAs, whereas the adult liver has anatomically isolated bile duct structures that separate blood flow and bile flow. However the role for these fetal BAs has been unknown. Here we report that bile acids support expansion of hematopoietic stem and progenitor cells (HSPCs) in the fetal liver and ex vivo. Since TUDCA is a rare component in human and mouse BAs, even in the fetal liver, we sought analogue(s) that similarly function as ER stress inhibitors. We identified that Taurocholic acid (TCA), one of the main components of fetal BA, and Tauro-alpha-muricholic acid (TαMCA) that is a rodent specific BA have a potential to reduce ER stress, similar to TUDCA. Mouse HSCs cultured with TCA or TαMCA in vitro for 2 weeks showed a robust increase in the reconstitution level compared to non-treated cells (14-fold, n=14, p<0.001 and 13-fold, n=9, p<0.05, respectively), which has comparable or even better potential to support HSC function than TUDCA. To study physiological roles of BA in ER stress reduction and HSC expansion in the fetal liver, an inhibitor of BA synthesis, GW4064 (an agonist of a nuclear receptor FxR that negatively regulates key enzymes in the BA biosynthesis, CYP7A1 and CYP8B1), was intraperitoneally injected into pregnant mice. E16.5 fetuses derived from GW4064-injected pregnant mice showed severe decrease in the number of HSPCs (0.40-fold, n=28-33, p<0.001) in the fetal liver, due to increased apoptosis triggered by elevated ER stress levels. Importantly, co-injection of TCA or Salubrinal (inhibitor of the ER stress-induced apoptosis signal) rescued the effects of GW4064 on cellularity of the fetal liver and levels of ER stress, confirming that the phenotype seen here is due to increased ER stress resulting from lowered levels of BA. Analyses of CYP27A1 knockout (KO) mice that have reduced BA synthesis observed decreased HSC number and increased ER stress in the fetal liver, whereas CYP8B1 KO mice that have increased Tα/βMCA synthesis instead of lack of TCA didn’t show any difference. These findings strongly suggest that fetal BA, particularly TCA and TαMCA, supports the expansion of HSPCs in the fetal liver and the ex vivo culture as chemical chaperones by lowering ER stress levels. Our findings propose a new role of bile acids in hematopoiesis as natural chaperones and provide a novel connection between hematopoiesis and fetal liver. Disclosures No relevant conflicts of interest to declare.

scholarly journals Notch-Mediated Expansion of Human Hematopoietic Stem and Progenitor Cells By Culture Under Hypoxia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Daisuke Araki ◽  
Stefan Cordes ◽  
Fayaz Seifuddin ◽  
Luigi J. Alvarado ◽  
Mehdi Pirooznia ◽  
...  
Keyword(s):  
Er Stress ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Human Adult ◽  
Hypoxic Conditions ◽  
Cd34 Cells ◽  
Human Hscs ◽  
Ex Vivo Culture ◽  
Cells Cultured

Notch activation in human CD34+ hematopoietic stem/progenitor cells (HSPCs) by treatment with Delta1 ligand has enabled clinically relevant ex vivo expansion of short-term HSPCs. However, sustained engraftment of the expanded cells was not observed after transplantation, suggesting ineffective expansion of hematopoietic stem cells with long-term repopulating activity (LTR-HSCs). Recent studies have highlighted how increased proliferative demand in culture can trigger endoplasmic reticulum (ER) stress and impair HSC function. Here, we investigated whether ex vivo culture of HSPCs under hypoxia might limit cellular ER stress and thus offer a simple approach to preserve functional HSCs under high proliferative conditions, such as those promoted in culture with Delta1. Human adult mobilized CD34+ cells were cultured for 21 days under normoxia (21% O2) or hypoxia (2% O2) in vessels coated with optimized concentrations of Delta1. We observed enhanced progenitor cell activity within the CD34+ cell population treated with Delta1 in hypoxia, but the benefits provided by low-oxygen cultures were most notable in the primitive HSC compartment. At optimal coating densities of Delta1, the frequency of LTR-HSCs measured by limiting dilution analysis 16 weeks after transplantation into NSG mice was 4.9- and 4.2-fold higher in hypoxic cultures (1 in 1,586 CD34+ cells) compared with uncultured cells (1 in 7,706) and the normoxia group (1 in 5,090), respectively. Conversely, we observed no difference in expression of the homing CXCR4 receptor between cells cultured under normoxic and hypoxic conditions, indicating that hypoxia increased the absolute numbers of LTR-HSCs but not their homing potential after transplantation. To corroborate these findings molecularly, we performed transcriptomic analyses and found significant upregulation of a distinct HSC gene expression signature in cells cultured with Delta1 in hypoxia (Fig. A). Collectively, these data show that hypoxia supports a superior ex vivo expansion of human HSCs with LTR activity compared with normoxia at optimized densities of Delta1. To clarify how hypoxia improved Notch-mediated expansion of LTR-HSCs, we performed scRNA-seq of CD34+ cells treated with Delta1 under normoxic or hypoxic conditions. We identified 6 distinct clusters (clusters 0 to 5) in dimension-reduction (UMAP) analysis, with a comparable distribution of cells per cluster between normoxic and hypoxic cultures. Most clusters could be computationally assigned to a defined hematopoietic subpopulation, including progenitor cells (clusters 0 to 4) and a single transcriptionally defined HSC population (cluster 5). To assess the relative impact of normoxia and hypoxia on the HSC compartment, we performed gene set enrichment analysis (GSEA) of cells within HSC cluster 5 from each culture condition. A total of 32 genes were differentially expressed, and pathways indicative of cellular ER stress (unfolded protein response [UPR], heat shock protein [HSP] and chaperone) were significantly downregulated in hypoxia-treated cells relative to normoxic cultures (Fig. B). When examining expression of cluster 5 top differentially expressed genes across all cell clusters, we observed a more prominent upregulation of these genes within transcriptionally defined HSCs exposed to normoxia relative to more mature progenitors (Fig. C, red plots). Hypoxia lessened the cellular stress response in both progenitors and HSCs, but the mitigation was more apparent in the HSC population (Fig. C, grey plots), and decreased apoptosis was observed only within the HSC-enriched cluster 5 (Fig. D). These findings are consistent with several reports indicating that HSCs are more vulnerable to strong ER stress than downstream progenitors due to their lower protein folding capacity. In conclusion, we provide evidence that ex vivo culture of human adult CD34+ cells under hypoxic conditions enables a superior Delta1-mediated expansion of hematopoietic cells with LTR activity compared with normoxic cultures. Our data suggest a two-pronged mechanism by which optimal ectopic activation of Notch signaling in human HSCs promotes their self-renewal, and culture under hypoxia mitigates ER stress triggered by the increased proliferative demand, resulting in enhanced survival of expanding HSCs. This clinically feasible approach may be useful to improve outcomes of cellular therapeutics. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ex Vivo Modeling of Hematopoietic Stem Cell Homing to the Fetal Liver

2020 ◽  
Author(s):  
Amina Mohammadalipour ◽  
Miguel F. Diaz ◽  
Sumedha Pareek ◽  
Pamela L. Wenzel
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Cell Homing ◽  
Stem Cell Homing

Recreating a Human Hematopoietic Stem Cell Niche in Vitro by Unique Stroma Cells from the First Trimester Human Placenta and Fetal Liver

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3568-3568
Author(s):  
Mattias Magnusson ◽  
Melissa Romero ◽  
Sacha Prashad ◽  
Ben Van Handel ◽  
Suvi Aivio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Placenta ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Embryonic Stem ◽  
Cell Types ◽  
First Trimester ◽  
Hematopoietic Stem ◽  
Human Hscs

Abstract Expansion of human hematopoietic stem cells (HSCs) ex vivo has been difficult due to limited understanding of their growth requirements and the molecular complexity of their natural microenvironments. To mimic the niches in which human HSCs normally develop and expand during ontogeny, we have derived two unique types of stromal niche cells from the first trimester human placenta and the fetal liver. These lines either support maintenance of multipotential progenitors in culture, or promote differentiation into macrophages. Impressively, the supportive lines facilitate over 50,000-fold expansion of the most immature human HSCs/progenitors (CD34+CD38-Thy1+) during 8-week culture supplemented with minimal cytokines FLT3L, SCF and TPO, whereas the cells cultured on non-supportive stroma or without stroma under the same conditions differentiated within 2 weeks. As the supportive stroma lines also facilitate differentiation of human hematopoietic progenitors into myeloid, erythroid and B-lymphoid lineages, we were able to show that the expanded progenitors preserved full multipotentiality during long-term culture ex vivo. Furthermore, our findings indicate that the supportive stroma lines also direct differentiation of human embryonic stem cells (hESC) into hematopoietic progenitor cells (CD45+CD34+) that generate multiple types of myeloerythroid colonies. These data imply that the unique supportive niche cells can both support hematopoietic specification and sustain a multilineage hematopoietic hierarchy in culture over several weeks. Strikingly, the supportive effect from the unique stromal cells was dominant over the differentiation effect from the non-supportive lines. Even supernatant from the supportive lines was able to partially protect the progenitors that were cultured on the non-supportive lines, whereas mixing of the two types of stroma resulted in sustained preservation of the multipotential progenitors. These results indicate that the supportive stroma cells possess both secreted and surface bound molecules that protect multipotentiality of HSCs. Global gene expression analysis revealed that the supportive stroma lines from both the placenta and the fetal liver were almost identical (r=0.99) and very different from the non-supportive lines that promote differentiation (r=0.34), implying that they represent two distinct niche cell types. Interestingly, the non-supportive lines express known mesenchymal markers such as (CD73, CD44 and CD166), whereas the identity of the supportive cells is less obvious. In summary, we have identified unique human stromal niche cells that may be critical components of the HSC niches in the placenta and the fetal liver. Molecular characterization of these stroma lines may enable us to define key mechanisms that govern the multipotentiality of HSCs.

Loss of PERK Signaling Results in Impaired Granulopoiesis in Transgenic Mice Expressing Mutant Ela2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 551-551
Author(s):  
Suparna Nanua ◽  
Jun Xia ◽  
Mark Murakami ◽  
Jill Woloszynek ◽  
Daniel C. Link
Keyword(s):  
Er Stress ◽  
Protein Misfolding ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Clinical Samples ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Granulocytic Differentiation ◽  
Disease Pathogenesis ◽  
Chemical Inducers

Abstract Abstract 551 Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by chronic neutropenia, a block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Approximately 50% of cases of SCN are associated with germline heterozygous mutations of ELA2, encoding neutrophil elastase (NE). To date, 59 different, mostly missense, mutations of ELA2 have been reported. A unifying mechanism by which all of the different ELA2 mutants disrupt granulopoiesis is lacking. We and others previously proposed a model in which the ELA2 mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Testing this (and other) models has been limited by the rarity of SCN and difficulty in obtaining clinical samples for testing. We previously reported preliminary findings of a novel transgenic mouse expressing a truncation mutation of Ela2 (G193X) reproducing a similar mutation found in some patients with SCN (2008 ASH abstract #314). We showed that the G193X Ela2 allele produced the expected truncated protein that was rapidly degraded. Surprisingly, basal and stress granulopoiesis were normal. We hypothesized that reduced expression of Ela2 in murine compared with human granulocytic precursors resulted in less delivery of misfolded mutant NE protein to the ER, attenuating UPR activation and preserving granulopoiesis in G193X Ela2 mice. Consistent with this hypothesis, only modest evidence of UPR activation was observed in G193X Ela2 granulocytic precursors, and these cells displayed increased sensitivity to chemical inducers of ER stress compared with wildtype granulocytic precursors. The UPR model of disease pathogenesis predicts that inhibition of the cellular pathways that handle misfolded proteins may sensitize G193X Ela2 cells to ER stress and result in impaired granulocytic differentiation. To test this prediction, we crossed G193X Ela2 mice with mice lacking protein kinase RNA (PKR)-like ER kinase (PERK); PERK is one of three major ER-resident proteins that sense ER stress and activate the UPR. Of note, homozygous loss-of-function mutations of PERK (EIF2AK3) are responsible for Wolcott-Rallison syndrome, which is characterized by infantile diabetes and neutropenia in approximately 50% of cases. Since PERK deficiency is embryonic lethal, we transplanted fetal liver cells from PERK-/-, PERK-/- × G193X Ela2, and wild type embryos into irradiated recipients. Complete donor engraftment was observed in all cohorts. Basal granulopoiesis was normal in mice reconstituted with PERK-/- cells. However, in the PERK-/- × G193X Ela2 chimeras, though blood neutrophil counts were normal, a significant reduction in bone marrow neutrophils was observed [6.01 × 106/femur ± 0.92 (PERK-/-) versus 3.14 × 106 ± 0.88 (PERK-/- × G193X Ela2); p < 0.001]. These data show that loss of PERK signaling combined with G193X Ela2 expression results in impaired granulopoiesis, providing new evidence in support of the UPR model of disease pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Activation of Notch Signaling Mediates Ex Vivo Expansion of Multilineage, Engrafting Murine Hematopoietic Progenitors From Embryonic Sources.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2321-2321
Author(s):  
Brandon K Hadland ◽  
Barbara Varnum-Finney ◽  
Irwin D. Bernstein
Keyword(s):  
Notch Signaling ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Signal Pathways ◽  
Mouse Bone Marrow ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Notch Activation

Abstract Abstract 2321 An important goal in the application of pluripotent stem cells (PSC) for therapeutic purposes is the derivation of hematopoietic stem and progenitor cells (HSPC) capable of efficient engraftment in vivo. Fundamental to achieving this goal is improved understanding of key signal pathways required to establish, maintain and expand HSPCs from embryonic sources. Ex vivo activation of Notch signaling in mouse bone marrow and human cord blood-derived HSC can facilitate expansion of rapidly engrafting multilineage progenitors, which has recently been translated for therapeutic purposes. In contrast, similar expansion of engrafting progenitors has not been successful from PSC. This prompted us to evaluate whether embryonic-derived HSPC have capacity to respond to ligand-induced Notch signaling ex vivo, and whether Notch activation could promote expansion of engrafting progenitors from these embryonic sources. We have examined the effects of ex vivo activation of Notch receptors by immobilized, exogenous Notch ligands on highly enriched populations of embryonic HSC and HSC precursors (pre-HSC) at various developmental stages. We find that activation of Notch by the ligand Delta1 within HSC/pre-HSC isolated from embryonic aorta-gonad-mesonephros (AGM) promotes expansion of progenitors with erythromyeloid colony forming potential and T/B-lymphoid potential in vitro, with concurrent expression of surface phenotypes resembling fetal liver-stage HSC. Furthermore, Notch activation in embryonic HSPC also mediates expansion of progenitors with rapidly engrafting myeloid and lymphoid capacity in irradiated mouse models. Our results demonstrate that embryonic stage HSPC have capacity to expand in response to Notch activation, and thus further studies comparing AGM- and PSC-derived hematopoietic precursors are needed to elucidate differences that may account for failure to expand HSPC from PSC. Disclosures: Bernstein: Seattle Genetics, Inc.: Consultancy.

A GATA-2-GPR65 Regulatory Module Controls Hematopoietic Stem Cell Generation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1176-1176
Author(s):  
Xin Gao ◽  
Tongyu Wu ◽  
Jamie Lahvic ◽  
Kirby D. Johnson ◽  
Erik A. Ranheim ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Rt Pcr ◽  
Employment Equity ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
G Protein Coupled

Abstract The generation of hematopoietic stem cells (HSCs) via endothelial-to-hematopoietic transition within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. Many questions remain unanswered regarding the molecular program in hemogenic endothelium that promotes the budding of hematopoietic cell clusters containing HSCs. We demonstrated that a deletion of a Gata2 cis-element reduced GATA-2 levels in the AGM and abrogated the capacity of hemogenic endothelium to generate HSCs. Consistent with the defective HSC generator, the mutant fetal livers were deficient in hematopoietic stem and progenitor cells (HSPCs). Using an ex vivo intact AGM culture system, we demonstrated that retrovirus-mediated GATA-2 expression in the +9.5-/- AGM rescues its hematopoietic defect. Thus, the reduced GATA-2 levels in the +9.5-/- AGM cause the HSC generation defect, and this rescue assay provides a unique system to decipher the downstream genetic network. To discover novel druggable regulators in the GATA-2 pathway to promote HSC generation, we profiled the expression pattern of all G-protein-coupled-receptors, which represent the most successful class of pharmaceutical targets, in the AGM using our RNA-seq dataset (+9.5+/+ vs. +9.5-/- AGM). This global GPCR analysis revealed four GATA-1 and GATA-2 co-regulated genes, Adora3, Gpr65, Ltb4r1, and Adora2b. Database mining revealed that only the Gpr65 expression pattern resembled that of Gata2. To evaluate GPR65 functions during HSC generation, we conducted an shRNA-based loss-of-function analysis in the AGM. While downregulating Gpr65 did not alter the abundance of the CD31+ c-Kit+ hematopoietic cell population, it significantly increased the CD31+ c-Kit+ Sca1+ HSC-containing cell population (1.4 fold, p<0.05), indicating that GPR65 suppresses HSC generation. To validate the involvement of GPR65 during the HSC generation process in vivo, we conducted a morpholino oligonucleotide (MO)-based loss-of-function study in zebrafish. In situ hybridization analysis revealed high Runx1/c-Myb expression (labeling definitive HSCs and progenitors) in 48% of embryos injected with Gpr65 MOs compared with 11% of wild type embryos. Consistent with the ex vivo AGM analysis, this increase in Runx1/c-Myb expression upon Gpr65 MO treatment suggests GPR65 is a negative regulator of HSC emergence in vivo. To dissect the molecular mechanism governing GPR65-suppressed HSC generation, we tested whether lowering Gpr65 levels altered the expression of key HSC regulators. Quantitative RT-PCR analysis revealed that downregulating Gpr65 by 60-70% in AGM CD31+ c-Kit- endothelialcells increased Gata2 mRNA by 2.9 fold (p<0.05), Gata2 primary transcripts by 3.9 fold (p<0.05), and elevated expression of the GATA-2 target gene Runx1 (2.9 fold, p<0.05). These results support a mechanism whereby GPR65-mediated Gata2 repression is an important determinant of GPR65-suppressed HSC generation. In addition to this important function in the AGM, Gpr65 knockdown studies in primary fetal liver HSPCs revealed GPR65 suppression of Gata2 transcription to the same magnitude as in the AGM. To determine if GPR65-mediated Gata2 repression requires the +9.5 site, we infected freshly isolated HSPCs from fetal livers heterozygous for the +9.5 site with retrovirus expressing shRNA targeting Gpr65. Quantitative RT-PCR with allele-specific primers revealed that Gpr65 knockdown significantly upregulates Gata2 primary transcripts from the wild type (3.1 fold, p<0.01), but not the 9.5 mutant, allele. These results establish a requirement of the +9.5 site for GPR65 to repress Gata2 transcription. As we reported that SetD8, the only enzyme known to monomethylate H4K20, represses Gata2 expression via the +9.5 site, we tested whether GPR65 represses Gata2 expression through SetD8. H4K20me1 ChIP revealed that downregulating Gpr65 significantly reduces H4K20me1 levels at the +9.5 site by 30% (p<0.005), suggesting that GPR65 repression of Gata2 transcription involves SetD8. Our studies indicate that a G-protein coupled receptor, GPR65, is negative regulator of HSC generation and establish a GATA-2-GPR65 Type Iincoherent feedforward loop that controls HSC generation, providing a foundation to develop new targets for expanding HSCs for transplantation therapies and a new druggable target to treat hematologic disorders. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

scholarly journals Ex Vivo Expansion of Functional Human UCB-HSCs/HPCs by Coculture with AFT024-hkirreCells

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Muti ur Rehman Khan ◽  
Ijaz Ali ◽  
Wei Jiao ◽  
Yun Wang ◽  
Saima Masood ◽  
...  
Keyword(s):  
Cell Line ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Critical Role ◽  
Ex Vivo Expansion ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Hematopoietic Growth Factors ◽  
Hematopoietic Stem

Kiaa1867 (human Kirre, hKirre) has a critical role in brain development and/or maintenance of the glomerular slit diaphragm in kidneys. Murine homolog of this gene, mKirre expressed in OP9 and AFT024 cells could support hematopoietic stem cells/hematopoietic progenitor cells (HSC/HPC) expansion in vitro. HKirre is also expressed in human FBMOB-hTERT cell line and fetal liver fibroblast-like cells but its function has remained unclear. In this paper, we cloned a hKirre gene from human fetal liver fibroblast-like cells and established a stably overexpressing hKirre-AFT024 cell line. Resultant cells could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than AFT024-control cells transformed with mock plasmid. The Expanded human umbilical cord blood (hUCB) CD34+cells retained the capacity of multipotent differentiation as long as 8 weeks and successfully repopulated the bone marrow of sublethally irradiated NOD/SCID mice, which demonstrated the expansion of long-term primitive transplantable HSCs/HPCs. Importantly, hkirre could upregulate the expressions of Wnt-5A, BMP4, and SDF-1 and downregulate TGF-βwith other hematopoietic growth factors. By SDS-PAGE and Western Blot analysis, a ~89 kDa protein in total lysate of AFT024-hKirre was identified. Supernatants from AFT024-hkirre could also support CD34+CD38−cells expansion. These results demonstrated that the AFT024-hKirre cells have the ability to efficiently expand HSCs/HPCs.

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