Generation Of Stem Cells Research Articles

Single Cell Transcriptomic Analysis of B-Cell Acute Lymphoblastic Leukemia (B-ALL) Reveals Correlation between Leukemia Cell State, Genetic Alterations and Clinical Outcome

I.I., A.G.X.Z, Q.G., L.G.P.: co-first authors; J.E.D., C.G.M.: co-senior authors Introduction: Genomic analyses of bulk ALL samples have improved our understanding of the genetic basis and risk stratification of B-ALL, but do not directly examine intratumor heterogeneity or enable inference of leukemia developmental state and cell of origin. Methods: We profiled 89 B-ALL samples by single-cell RNA-seq (scRNA-seq) (10X Genomics 5'v2) and compared them to a scRNA-seq reference map of normal human B-cell development. Results: Analysis of heterogeneity of inferred DNA copy number alterations at single cell level showed that aneuploid ALL with near haploidy and hyperdiploid harbor chromosomal losses or gains in all blasts, consistent with early, synchronous origin rather than sequential accumulation. A subset of TCF3::PBX1 samples harbored clones with progressive overexpression of genes surrounding PBX1 which scWGS confirmed caused by accumulation of DNA gains. Extensive intra-sample heterogeneity was observed by non-negative matrix factorization and driven by five consensus gene expression signatures: cell cycle (S; G2/M), metabolism, differentiation and inflammation. Signature composition refined leukemic subtyping; for example, within DUX4-r B-ALL two groups were discerned: one with higher inflammation score, expression of stem cell genes and worse outcome and one with lower inflammation score and expression of Pro-B related genes. To understand variation in developmental states among B-ALL blasts, we first developed an atlas of human B cell development. Marker genes elucidated from bulk RNA-seq on purified human HSC, MPP, LMPP, MLP, CLP, Pre-Pro-B, Pro-B, Pre-B, and B populations guided the development of a new scRNA-seq atlas of human B cell development comprising 130,085 cells spanning 90 fetal, pediatric, and adult samples from 8 studies (Fig 1A). Unexpectedly, we found that human CLPs retain transcriptional programs governed by CEBPA; in vitro and in vivo functional assays validated the capacity of CLPs for extensive myeloid differentiation prior to lineage restriction at the Pro-B stage. We next mapped our 89 B-ALL sc samples to precise cellular states along B cell development and clustered samples by leukemia cell composition. As expected, most samples exhibited similarity of GEP to that of pro-B cells, and this was most pronounced among hyperdiploid samples. High pre-B abundance was observed in MEF2D-r and TCF3::PBX1, while an HSC/MPP/LMPP enriched cluster encompassed ZNF384-r and a subset of DUX4-r B-ALL. Notably, one ZNF384-r B-ALL patient with LMPP involvement at diagnosis retained these LMPP-like cells at relapse despite a lineage switch to AML. Finally, we identified a group of patients with high early lymphoid (MLP, CLP, Pre-Pro-B) abundance representing a subset of Ph+ and KMT2A-r samples (Fig. 1B). We next utilized marker genes from each B-ALL cell state to estimate their relative abundance within a bulk RNA-seq cohort of 2046 B-ALL patients. In addition to validating these associations between genomic class and leukemia cell state, we identified age-dependent patterns of B-ALL state involvement wherein early lymphoid involvement is highest in infancy and adulthood while Pro-B involvement is highest in childhood. In childhood, high-risk disease is associated with higher early lymphoid ( P=4.9e-12) and lower Pro-B ( P=0.0085) abundance. Further, patients with residual disease (RD) levels above 1% are enriched for higher upfront early lymphoid abundance ( P=1e-7) and lower Pro-B ( P=4.1e-5) and Pre-B ( P=2.2e-11) abundance compared to patients with RD < 0.01%. Finally, B-ALL cell state improved resolution of previously reported transcriptional heterogeneity within defined genomic classes. Early lymphoid abundance discerns between two subtypes within Ph+ (Kim Nat Gen 2023) with AUC=0.953 ( P=2.4e-9), as well as two subtypes within KMT2A-r (Brady Nat Gen 2022) with AUC=0.996 ( P=1.1e-6). Notably we find that KMT2A::AFF1 fusions produce early lymphoid-enriched disease compared to KMT2A::MLLT3 ( P=0.0024) and KMT2A::MLLT10 ( P=0.0037), wherein the latter two fusions exhibit Pre-B enrichment ( P=0.025 and P=0.0037, respectively). Conclusions: Understanding variation in transcriptional programs and developmental states of B-ALL blasts by sc transcriptome refines existing clinical and genomic classifications and provides novel prognostic markers.

UM171 Enhances Fitness and Engraftment of Gene Modified Hematopoietic Stem Cells from Sickle Cells Disease Patients

Sickle cell disease (SCD) is one of the most common genetic blood diseases. Autologous hematopoietic stem cell gene therapy using lentiviral vectors is an effective therapeutic strategy for SCD. However, the occurrence of post gene therapy myelodysplastic syndrome and acute myeloid leukemia not related to vector insertion has suggested an underlying genetic risk or acquisition of damage during ex vivo culture or from proliferative stress associated with reconstitution. Here, we aimed to improve the transduction efficiency, stem cell fitness and reconstitution of SCD CD34+ cells transduced with a lentiviral vector (LV) containing BCL11A shmiR currently in clinical trials (Esrick et al. NEJM, 2021, NCT03282656), to minimize culture-induced stress and reduce genomic damage during ex vivo culture. UM171, an HSC self-renewal pyrimidoindole derivative agonist, has been shown to expand HSCs and enhance multilineage blood cell reconstitution in mice. We examined the effect of addition of UM171 during ex vivo transduction on HSCs from SCD patients. Culture of SCD CD34+ HSC in HSC expansion conditions with UM171 increased the proportion of CD34+CD133-CD45RA-CD90+ long term HSCs (LT-HSCs), from 1.1% to 4.1% (N = 3, P < 0.001), and the absolute number of LT-HSCs increased 4-fold (N = 3, P < 0.001). Treatment with UM171 also significantly enhanced the transduction efficiency of BCL11A shmiR containing lentiviral vector (LV-BCL11A) in SCD CD34+ cells from 0.7 vector copies/diploid genome (VCN) to 1.0 VCN (N = 5, P < 0.001), and significantly decreased apoptosis of stem cells from 22.5% to 14.3% (N = 3, P < 0.001), and DNA damage as measured by γ-H2AX from 10.6% to 6.6% (N = 3, P < 0.001). Given these results, we assessed the engraftment capability and clonality of SCD CD34+ transduced in the presence or absence of UM171 in NBSGW mice. UM171 increased the human CD45 engraftment of mice measured in BM at 16 weeks post-transplantation from 32.5% to 69.4% in hCD45+ BM cells (N = 6, P < 0.01) and enhanced the engraftment of transduced in SCD CD34+-derived CD45+ cells from 11.2% to 18.2% in gene-marked BM cells (N = 6, P < 0.01), while maintaining multilineage differentiation in this model. Barcode analysis revealed the engraftment of 500-1800 unique barcodes with a high degree of clonal diversity and a trend towards increased polyclonal engrafting populations in transplanted mice. To assess the effect of UM171 more rigorously on functionally defined HSCs, we performed a competitive transplantation assay using CD34+ cells transduced in the presence vs absence of UM171. Analysis of BM obtained from the mice at 16 weeks post-transplantation showed that cells transduced in the presence of UM171 consistently outcompeted those transduced under control conditions, 62.6% in gene-marked erythroid cells of UM171 treated cells compared with 37.4% of control cells (N = 9, P < 0.001), and UM171 maintains LV-BCL11A induced γ-globin expression in erythroid cells. In summary, a short-term exposure of SCD CD34+ PB cells to UM171 enhances stem cell fitness. Implementing these findings in clinical gene therapy protocols may improve the efficacy and sustainability of gene therapy and generate new opportunities in the field of gene editing.

Α-Globin Lentiviral Vectors for Hematopoietic Stem Cell Gene Therapy of α-Thalassemia

α-thalassemia is an inherited blood disorder caused by mutations in α-globin genes (HBA1 and HBA2) resulting in the reduction of α-globin chains, the subunit, along with β-globin chain, constituting adult hemoglobin (α 2β 2). While a reduction of α-globin expression of 40% presents asymptomatic phenotypes, expression levels decreasing to <20% or an α/β-globin mRNA ratio of <0.2, causes severe anima. Treatment for severe cases of α-thalassemia entails lifelong, biweekly blood transfusions with daily chelation therapy. While these therapies enable patients to live into mid- to late-adulthood, they continue to engender serious clinical manifestations, such as iron toxicity. To answer this clinical need, our laboratory has developed a stem cell gene therapy in which functional copies of α-globin gene integrate into the genome of patient's hematopoietic and progenitor stem cells (HSPCs) by lentiviral vectors (LVs) to normalize the globin chain imbalance and restore hemoglobin function in derived red blood cells (RBCs). The design of our α-globin LVs (AGLVs) is based on GLOBE β-globin LV utilized in a clinical trial for β-thalassemia (NCT02453477), which has achieved transfusion reduction and independence in patients with transfusion-dependence β-thalassemia major. We have constructed a series of short proviral length AGLVs for optimized titer production, HSPC infectivity, and lineage-specific α-globin gene expression. Twelve AGLVs varying in gene and regulatory element compositions were constructed and assessed for raw titer yields and characterized for gene transfer efficiency, mRNA expression and hemoglobin production in a α-globin knockout (KO) human erythroid cell line. Successfully, all AGLVs confer high raw titers ~ 1e7 TU/mL and enabled the production of adult hemoglobin (HbA). We identified two optimal AGLVs for further characterization in human primary HSPCs: 1) A2-UV for yielding highest raw titers and gene transfer efficiency, and 2) A2-Globe for producing the most α-globin mRNA. Both A2-UV and A2-Globe LVsharbor HBA2, are regulated by the β-globin promoter and enhanced by the core or large β-LCR enhancer region, respectively. To assess candidate AGLVs in human HSPCs, AGLVs were tagged at the transcription level to enable identification and quantification of vector-derived α-globin mRNA proceeding HSPCs transduction anderythroid differentiation. A2-UV demonstrated optimum CD34 + infectivity and gene transfer, and A2-Globe expressed a high ~30% of α-globin mRNA per total β-globin per vector copy number (VCN), achieving levels of one endogenous α-globin gene (~25% per total β-globin). Current studies examined A2-UV and A2-Globe in α-thalassemia major patient HSPCs - lacking all four α-globin genes - harboring a lethal homozygous 20kb deletion of HBA1 and HBA2, known as SEA –/– mutation. Patient HSPCs were transduced and differentiated into RBCs. A high CD34+ infectivity and gene transfer was obtained by both candidate vectors, without utilization of transduction enhancers. Furthermore, a correction of α/β-globin mRNA ratio of ~0.7 was obtained while retaining a low VCN (<3).RBCs were then analyzed at the protein level . Chromatograms of hemoglobin tetramers revealed that HbA represented 98% of all hemoglobin, compared to 0% in non-transduced patient RBCs. Single globin chain chromatograms successfully demonstrated α/β-globin chain ratio of ~0.4 or higher and successfully demonstrated that a low average of copies per cell (~3) restored 60% of adult hemoglobin when normalized to healthy donor RBCs. These data demonstrate α/β-globin correction and hemoglobin restoration in α-thalassemia patient cells with the most severe mutation. Three assays were conducted on patient cells, all with similar and highly encouraging results for disease amelioration in severe cases of α-thalassemia. Overall, this project is paving the way for the first development of a curative gene therapy approach for α-thalassemia.

A Pro-Inflammatory Gene Signature Characterizes a Better Risk Aged AML Patient Group in ECOG-ACRIN Cancer Research Group's Clinical Trial E3999

Background: Acute myeloid leukemia in patients over the age of 60 (aAML) is associated with poor prognosis. aAML patients are the majority diagnosed but are underrepresented in large molecular studies. We have identified two clinical subgroups of aAML patients with distinct survival outcomes (low-risk and high-risk) in ECOG-ACRIN Cancer Research Group's clinical trial E3999 (NCT00046930). We hypothesize that these two groups are characterized by distinct biological mechanisms that contribute to leukemogenesis. We also hypothesize that the low-risk group harbor molecular and phenotypic signatures characteristic of known better-risk AMLs. Methods: Diagnostic specimens were obtained from patients enrolled in NCT00046930. Disease enriched cells were isolated by negative selection of lymphocytes (magnetic beads). Flow cytometry was used to characterize the blast population. Bulk RNA-sequencing was performed on 220 blast enriched samples and 10 age matched normal controls (CD34+ bone marrow cells). Transcriptional differences were determined using DESeq2. Differentially expressed genes (DEGs) were identified as those with an absolute log2 fold change greater than 1 and q less than 0.05. Gene set enrichment analysis (GSEA) was performed against the MSigDB databases. Upstream regulators associated with gene expression changes were identified using Ingenuity Pathway Analysis (IPA). DESeq2 was used to identify genes that were differentially associated with overall survival times between the risk groups. Enrichment for transcription factor gene targets was calculated using EnrichR based on the CHEA Transcription Factor Target Dataset. Cytokines were quantified in a subset of patient serum samples using a Luminex assay. Results: We identified 2314 DEGs between the low-risk and high-risk aAMLs with high-risk as the baseline. The upregulated genes in the low-risk aAMLs were positively enriched for gene signatures derived from: 1. Favorable risk AML ( NPM1 mutated), 2. M4 and M5 AML patients, 3. Differentiated hematopoietic cells and 4. Inflammatory response pathways. On the contrary, upregulated genes in the high-risk aAMLs were enriched for gene signatures identified in hematopoietic and leukemic stem cells and known poor-risk AMLs (for example: EVI1 fusion). Accordingly, immunophenotypic data from flow cytometry results gated on the blast populations validated differences in the surface expression of hematopoietic cell markers between the two groups. Next, we sought to determine upstream regulators associated with the DEGs using IPA. We identified 107 potential upstream regulators that associated with the changes in gene expression observed between the low- and high-risk groups (87 activated and 20 inhibited). The results suggested that several pro-inflammatory cytokines (ex. tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ)) were predicted activators in the low-risk aAML group. Accordingly, we detected significantly higher levels of TNFα and IFNγ in the serum levels of a subset of low-risk aAML patients. Finally, we assessed for genes whose expression significantly associated with overall survival. Interestingly, in agreement with a previously proposed inflammation score (iScore), we identified higher iScore associated with the high-risk group. Of note, the upstream regulators identified do not overlap with those contributing to the calculation of iScore. Additionally, longer overall survival times were associated with higher levels of expression of genes associated with heme metabolism and erythroblast differentiation in the low-risk aAML patient group. Conclusions: Our results suggest that the low-risk aAML patients were characterized by a more mature hematopoietic cell gene expression signature and cell surface markers as well as a pro-inflammatory state. Our study supports the possibility that pro-inflammatory cytokines and/or signaling could mediate AML cell survival in the low-risk aAML patient group. The high-risk aAML patients were characterized by a more primitive hematopoietic and leukemic stem cell gene expression signature. Collectively, we propose that differences in the underlying molecular and/or immune-based mechanisms could contribute to leukemogenesis in the two risk groups. These findings suggest that distinct therapeutic approaches may need to be considered to improve clinical outcomes in the two aAML risk groups.

Purine Metabolism Modulates Leukemia Stem Cell Maintenance in MLL-Rearranged Acute Leukemia

Acute myeloid leukemia (AML) represents the most prevalent hematopoietic malignancy driven by leukemia stem cells (LSCs) and remains challenging to treat in adult patients. MLL rearrangement results in a genetically unique subtype of AML characterized by chromosomal translocations involving the MLL gene and over 80 fusion partners, including AF9 as one of the most common partners. The oncogenic MLL fusion proteins interact with multiple transcription and chromatin regulators, such as Menin, to induce leukemogenic stem cell gene programs and transform hematopoietic stem and progenitor cells. Although significant progress has been made in understanding the metabolic dysregulation in acute leukemia, such as alterations in glycolysis, energetic metabolism, amino acid metabolism, and fatty acid metabolism, the metabolic factors that modulate MLL fusion oncogenic transcriptional activity in AML remain incompletely elucidated. To identify novel metabolic vulnerabilities in MLL-rearranged AML, we performed a whole genome CRISPR dropout screen and profiled the metabolites of murine LSCs of MLL-AF9 murine model. This analysis revealed the purine biosynthesis pathway is essential for MLL-rearranged leukemia and that MLL-rearranged LSCs are enriched in metabolites of the purine biosynthesis pathway. Isotypoe incorporation experiments using [U- 13C]glucose demonstrated a high purine biosynthetic rate in the LSCs compared to bulk leukemia cells. Accordingly, human acute myeloid leukemia stem cells express high levels of the purine biosynthetic genes. Furthermore, we found that MYC, a well-known downstream target of MLL-AF9, mediates the enhanced purine synthesis in LSCs. These results demonstrate enhanced purine biosynthetic pathways in LSCs. We utilized genetic and pharmacological (mycophenolate mofetil, MMF, a purine biosynthesis inhibitor of IMPDH) approaches to target the enhanced purine metabolism and found inhibition of the purine synthesis pathway promotes myeloid differentiation of both murine and human LSCs. Metabolomics analysis reveals a remarkable reduction of purine metabolites. Supplementing the LSCs with guanosine, an intermediate metabolite of the purine biosynthesis pathway, substantially rescued the differentiation. Importantly, treatment of MLL-AF9-driven murine leukemia mice with MMF impaired LSCs function, prolonged leukemia survival, and reduced leukemia burden in the peripheral blood and bone marrow, while causing minimal effects on normal hematopoiesis. These results demonstrate the reliance on enhanced purine metabolism in LSCs maintenance. Previous work has shown that the purine biosynthesis pathway is essential for rRNA transcription in the nucleolus, which could be inhibited by CX-5461. To verify the essentiality of the rRNA transcription in MLL-rearranged LSCs, we treated MLL-AF9-induced murine LSCs cells with CX-5461 and found CX-5461 promotes myeloid differentiation of LSCs. Moreover, we found that the myeloid differentiation induced by MMF or CX-5461 is associated with reduced chromatin occupancy of the MLL-AF9 complex, especially Menin, and downregulated expression of MLL-AF9 target genes, such as Meis1, Hoxa9 and Myc, suggesting a regulatory role of nucleolar rRNA transcription on MLL-AF9 oncogenic gene expression program. We are currently focusing on the synergistic effects of targeting purine metabolism and Menin inhibition on AML suppression. Altogether, our findings reveal that purine metabolism maintains nucleolar rRNA transcription homeostasis to modulate MLL fusion complex-induced leukemogenic transcriptional activity in LSCs. The enhanced purine metabolism emerges as a crucial dependency for LSCs, providing potential targets for novel therapeutic strategies in treating MLL-rearranged leukemia.

Polycom Group Protein Mel18 Inhibits Hematopoietic Stem Cell Self-Renewal through Repressing the Expression of Genes Important for Cellular Senescence and Proliferation

Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1), that act in concert to initiate and maintain stable gene repression. Bmi1 and Mel18 are two major homologs of the PCGF subunit within the canonical PRC1 complex. While Bmi1 is a positive regulator of hematopoietic stem cell (HSC) and leukemia stem cell (LSC) self-renewal, the role of Mel18 in normal and malignant hematopoiesis is not fully understood. To further determine the role of Mel18 in hematopoiesis, we have generated Mel18 conditional knockout mice ( Mel18 f/f-Mx1Cre +). Acute deletion of Mel18 in the hematopoietic compartment did not affect the frequency and survival of hematopoietic stem and progenitor cells (HSPCs). To determine the impact of Mel18 deficiency on HSPC proliferation, we performed serial replating assays and found that loss of Mel18 increases the replating potential of HSPCs in vitro. To determine the role of Mel18 in HSC self-renewal, we first performed serial bone marrow (BM) transplantation assays and found that Mel18 null BM cells show increased engraftment in both primary and secondary transplantation assays. Limiting dilution transplantation showed that there were more functional HSCs in the BM of Mel18 knockout mice. We then performed HSC transplantation assays and found that loss Mel18 enhances HSC self-renewal. Thus, we demonstrated that Mel18 inhibits HSC self-renewal and decreases the number of functional HSCs in vivo. To understand the mechanism by which Mel18 inhibits HSC self-renewal and proliferation, we performed RNA-seq using Mel18 +/+ and Mel18 -/- HSCs. We then performed gene set enrichment analysis (GSEA) to identify potential Mel18 target genes important for HSC behavior. We found that HSC gene signatures are enriched in Mel18 -/- HSCs. We also found that leukemia stem cell gene signatures and CBF-mutant-AML genes are significantly enriched in Mel18 -/- HSCs, suggesting that loss of Mel18 may prime normal HSCs for leukemic transformation. Notably, our RNA-seq assay showed that senescence-related genes, including S100a9 and S100a8, were down-regulated in Mel18 -/- HSPCs. Importantly, loss of Mel18 significantly decreased the number of senescent HSPCs. To understand how Mel18 regulates gene transcription, we performed ATAC-seq assays in Mel18 +/+ and Mel18 -/- HSPCs. Mel18 null HSPCs showed decreased chromatin accessibility near both S100a8 and S100a9 genes. We confirmed that both S100a8 and S100a9 are downregulated in Mel18 -/- HSPCs compared to Mel18 +/+ HSPCs. Given that cell cycle arrest is a common feature of cellular senescence, we also examined the expression of cell cycle related genes in Mel18 -/- HSPCs. The expression of cell cycle regulators, including CDK4 and CCND2, is increased in Mel18 -/- HSPCs. Mel18 can bind to a specific DNA sequence, 5‘-GACTNGACT-3‘, thereby repressing the expression of target genes. Mel18 directly binds to the CCND2 gene locus as revealed by ChIP-seq assays in embryonic stem cells, suggesting that Mel18 may directly repress CCND2 expression in HSPCs. In summary, we demonstrate that Mel18 inhibits HSC self-renewal via repressing the expression of genes that are important for cellular senescence and proliferation.

Forward Genetic Screen Implicates Drivers of Leukemic Progression in a Novel Model of Trp53 R270H myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure and a highly variable clinical course. The most catastrophic complication of MDS is transformation to secondary acute myeloid leukemia (sAML). Notably, mutations in TP53 confer the single highest risk of transformation to sAML and death. However, some patients with TP53 mutated MDS do not develop sAML, suggesting that additional genetic events cooperate with TP53 mutations to transform MDS to sAML. Understanding the mechanisms of transformation of MDS to sAML could provide targets for therapeutic intervention. To model the genetics of MDS, we crossed mice bearing Trp53 R270H ( Trp53 is the murine TP53 gene) and deletion of genes syntenic with human chromosome 5q (del(5q)). To discover how additional mutations contribute to disease progression, we utilized Sleeping Beauty (SB) transposon mutagenesis in Trp53 R270H/del(5q) mice. SB transposase mobilized SB mutagenic T2/Onc transposons which randomly insert within the genome. T2/Onc transposons are designed to induce gain or loss of function alterations depending on the site and orientation of insertion with respect to targeted genes. We used the Mx1-Cre transgene to activate SB transposase and T2/Onc transposition in hematopoietic progenitors. Trp53 R270H anddel(5q)(or cytogenetically normal, CN) mice were crossed to SB mice to generate donor mice of the following genotypes: Trp53 R270H/del(5q)/ SB, Trp53 R270H/CN /SB, Trp53 WT/del(5q)/ SB, Trp53 WT/CN/ SB mice, and mice without SB transposition, (no transposition, NT: Trp53 R270H/del(5q) /NT). Bone marrow cells were transplanted into recipients, and SB insertional mutagenesis was activated using pI-pC to activate Cre. Mice receiving Trp53 WT/CN/ SB bone marrow developed more frequent T-cell leukemia (n=3/10) than myeloid leukemia (n=1/10). In contrast, mice receiving Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB bone marrow developed predominantly myeloid leukemia (n=14/28) more commonly than T-cell leukemia (1/28). Mixed phenotype leukemia was seen in 7/28 of these mice. Together, these data demonstrate a strong bias towards myeloid disease in SB-mutagenized Trp53 R270H bone marrow. To identify genes with SB insertions, we performed RNA sequencing to detect SB T2/Onc transposon-endogenous genefusion transcripts. Among Trp53 WT/CN/ SB leukemias, the most common recurrent SB fusions involved Notch1 and Ikzf1 as has previously reported for SB-associated T-cell leukemias . Among Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB leukemias, the most common recurrent SB-fusions involved Erg, Eras and Il2rb with Erg fusions detected 85% of Trp53 R270H leukemias (n=17/20). SB inserted upstream of Erg promoter indicating that these fusions likely upregulate expression of Erg. Indeed, Erg levels are significantly higher in leukemias that express SB-Erg fusions relative to leukemias that do not (p<0.0023). ERG is not recurrently mutated in human AML, but the ERG gene locus is commonly amplified, especially TP53 mutant AML. ERG is known to support normal hematopoietic stem cell self-renewal. Notably, Erg-insertions were also detected in a model of MDS expressing stabilized cyclin E with SB-mediated progression to erythroleukemia (Loeb 2019). Using gene set enrichment analysis, we found that hematopoietic stem cell and leukemic stem cell signatures are enriched in Erg-SB fusion leukemias. In our analyses of two independent data sets (TCGA and BEAT AML), stem cell signatures are also among the most highly enriched pathways in human AMLs expressing high ERG levels. Furthermore, in a human AML single cell RNA sequencing dataset (van Galen 2019), we found that ERG expression is highest in AML cells with the most immature stem and progenitor-like features. Together, these findings implicate a role for ERG as a driver of progression of MDS to AML by enhancing aberrant self-renewal. In summary, we present a novel murine model of Trp53/del(5q) MDS. In this model, Erg upregulation is associated with progression to AML and upregulation of leukemia stem cell gene expression profiles. These data implicate ERG as a major contributor to progression of MDS to secondary AML in the setting of mutant p53. Understanding the mechanisms of disease progression and self-renewal in myeloid malignancies with p53 mutations is critical to define effective therapeutic strategies in these rapidly fatal, treatment resistant diseases.

MPN Transformation Is Characterized By Heterogeneous Shifts in Lineage Character

Introduction: The Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are characterized by the clonal proliferation of differentiated hematologic cells. However, there is a limited understanding of how perturbations at the transcriptional level may contribute to accelerated or blast-phase disease, together referred to here as transformation. Here we characterize transcriptomic characteristics and cell type composition of MPNs along the transition to transformed disease. Methods: RNAseq and clinical data were collected on 261 patients who were part of BeatAML, including those with MPN, de-novo AML, and transformed MPN. This was combined with data from 56 transformed MPN patients seen at MSK, 11 of whom also had paired chronic-phase data available. Differential expression analysis was performed with DESeq2; both GSEA and GSVA were used to evaluate gene-set enrichment and pathways of biological relevance. A custom RNA-seq lineage deconvolution algorithm was used to estimate hematopoietic stage proportions and was used for unsupervised hierarchical clustering of cases. Kaplan-Meier methods were used to estimate survival among transformed MPN lineage subtypes. Results: The initial analysis included 207 de novo AML cases and 75 MPN-BP cases from BeatAML and MSK on which RNAseq and molecular data were integrated across cohorts. Unsupervised analysis was performed using hierarchical clustering applied to batch corrected, variance stabilized RNA-seq count data. Transformed MPN cases were enriched in 2 of 6 clusters (N = 10, 33.3%; N = 36, 54.5%). Notably, the molecular characteristics in these clusters are varied, suggesting that multiple genomic perturbations may lead to similar pathways of transformation. Gene set variation analysis (GSVA) demonstrated a coalescence of transcriptomic perturbation into specific pathways for transformed MPNs, such as hematopoietic stem cell gene sets (q-value = 0.00018). On the contrary, AML cases typically demonstrated upregulation in later stages of hematopoiesis, such as monocytes (q-value =0.00016). In evaluating specific genes differentially expressed between transformed MPNs compared to de novo AML and tAML patients, microRNA MIR29B1 was particularly upregulated in MPN-BP (logFC = 9.52, q < 0.01), while the proto-oncogene CT45A1 had decreased expression in transformed MPN compared to other AMLs (logFC = -7.47, q < 0.01). Paired GSEA of the 11 patients with myelofibrosis MF and transformed disease showed that transformed disease was enriched for CD34-positive HSCs (NES = 2.64, q-value < 0.0001) and E2F transcription factors (NES 2.66, q-value < 0.0001), as well as doxorubicin resistance (NES 2.40, q-value < 0.0001). Given enrichment of the CD34 positive HSC geneset, we performed our RNA-seq based lineage deconvolution on each of the paired samples ( Figure 1). MPNs appeared to transform in a heterogenous fashion reminiscent of the biological diversity in AML. In aggregate, transformation was associated with decreased monocytic (p = 0.002), natural killer (NK) (p = 0.042) character and expansion of common lymphoid progenitor character (p = 0.022). Lineage composition changes varied, with certain samples showing HSC expansion (ID: 54910, ID: 46567) and others exhibiting LMPP expansion (ID: 29485, 2456). Figure 2 defines seven distinct clusters of transformed MPN patients, driven by their lineage components. While these clusters are transcriptionally distinct, we again see that they are not defined by their genomic or cytogenetic alterations. We evaluated for a difference in overall survival (OS) between each cluster and intermediate-risk de novo AML. We found significantly worse OS among patients in three MPN-BP clusters - MPP (p =0.008), LMPP (p = 0.038), and GMP (p < 0.001) - represented as clusters 2, 5, and 7 in Figure 2. Compared to intermediate risk AML, transformed MPNs had significantly worse OS (HR: 1.79 95% CI: 1.18, 2.71, p = 0.0057). Discussion: To our knowledge, this is the first ever description of the transcriptional characteristics of MPN-BP, its comparison to de novo AML, and the first paired comparison of transcriptional and lineage characteristics for patients with chronic-phase MPNs that subsequently transformed. Our study provides valuable insights into the transcriptomic landscape of transformed MPN including both accelerated and blast phase disease, and draws novel contrasts to de novo AML.

Automatic generation of alloreactivity-reduced donor lymphocytes and hematopoietic stem cells from the same mobilized apheresis product

IntroductionIn vitro or in vivo depletion of alloreactive T cells can facilitate haplo-identical hematopoietic stem cell transplantation (HSCT). Very satisfactory transplant outcomes were thus reported for TCRαβ/CD19-depleted hematopoietic stem/progenitor cell (HSPC) grafts. The current semi-automatic manufacturing process on the CliniMACS Plus, although robust, still requires a significant amount of manual labor to be completed. Towards advancing and further facilitating large scale cell processing, a new TCRαβ/CD19 depletion module combined with the previously described CD45RA depletion module (to serve as allo-reactivity attenuated donor lymphocyte infusion) was established on the CliniMACS Prodigy.MethodsWe evaluated six apheresis products from G-CSF-mobilized volunteer donors which were split automatically by the Prodigy, one portion each depleted of CD45RA+ or of TCRαβ+ and CD19+ cells. We investigated critical quality attributes for both products. Products were assessed for recovery of HSPCs and mature subsets, as well as depletion efficiency of targeted cells using flow cytometry. Effects of apheresis and product age post 48 h storage at 2–6 °C as well as freeze-thawing on product viability and recovery of WBC and HPSCs were assessed by flow cytometry.ResultsTen sequential automatic processes were completed with minimal hands-on time beyond tubing set installation. Depletion efficiency of CD45RA+ resp. TCRαβ+ and CD19+ cells was equivalent to previous reports, achieving mean depletions of 4 log of targeted cells for both products. HSPC products retained TCRγδ+ and NK cells. 48 h storage of apheresis product was associated with the expected modest loss of HSPCs, but depletions remained efficient. Depleted products were stable until at least 72 h after apheresis with stem cell viabilities > 90%. Freeze-thawing resulted in loss of NK cells; post-thaw recovery of viable CD45+ and HSPCs was > 70% and in line with expectation.ConclusionThe closed, GMP-compatible process generates two separate medicinal products from the same mobilized apheresis product. The CD45RA-depleted products contained functional memory T cells, whereas the TCRαβ/CD19-depleted products included HSPCs, TCRγδ+ and NK cells. Both products are predicted to be effectively depleted of GVH-reactivity while providing immunological surveillance, in support of haplo-identical HSCT.Graphical

Generation of four gene-edited human induced pluripotent stem cell lines with mutations in the ATM gene to model Ataxia-Telangiectasia

Ataxia-Telangiectasia (A-T) is an autosomal recessive multi-system disorder caused by mutations in the ataxia-telangiectasia mutated (ATM) gene, resulting, among other symptoms, in neurological dysfunction. ATM is known to be a master controller of signal transduction for DNA damage response, with additional functions that are poorly understood. CRISPR/Cas9 technology was used to introduce biallelic mutations at selected sites of the ATM gene in human induced pluripotent stem cells (hiPSCs). This panel of hiPSCs with nonsense and missense mutations in ATM can help understand the molecular basis of A-T.

Embryonic Stem Cell Gene Targeting and Chimera Production in Mice.

Producing a custom gene mutation in embryonic stem (ES) cells, whether through homologous recombination or CRISPR-Cas gene editing, is the first step along the way to getting the mutation into live mice. However, there are a number of additional steps along the way, each presenting technical challenges. Here, we provide a guide for troubleshooting when the results are not as expected and to distinguish technical problems from possible biological effects of the mutation. From the isolation of clonal lines of targeted ES cells through the production of ES cell chimeras with the targeted ES cell clone, we discuss common technical problems and their most likely causes and solutions. We also provide guidance for situations where the mutation has a phenotype in the form of a dominant effect on ES cells or in chimeras.

Allogeneic Hematopoietic Cell Transplantation for Vexas Syndrome: Results of a Multicenter Study from the Chronic Malignancies and Autoimmune Disease Working Parties of the EBMT

VEXAS is a new entity encompassing a variety of clinical manifestations spanning from autoimmune/auto-inflammatory to hematologic conditions including MDS, plasma cell dyscrasias, and recurrent thrombosis. Somatic mutations of the X-linked UBA1 gene in hematopoietic stem and progenitor cells constitute the molecular underpinnings of VEXAS, typically occurring in men during the 6 th decade of life. Over the last 2 years since its first description, VEXAS has elicited wide medical interest resulting in the identification of pleomorphic clinical phenotypes, which make this syndrome a “big masquerader” and a clinical diagnostic challenge. Treatment strategies aim at suppression of the UBA1-mutant clone (e.g., with azacitidine or allo-HCT) or blockade of the downstream pleiotropic effects of hypercytokinemia. However, no treatment guidelines exist to date, and patients may manifest life-threatening inflammatory symptoms frequently refractory to many therapies. As few literature reports have described the successful use of transplant, we here report the outcomes of patients with VEXAS in a multicenter EBMT registry-based study. Leveraging a multidisciplinary liaison of EBMT and key rheumatology centers across Europe, we disseminated a survey to gather further information on patients with VEXAS undergoing allo-HCT to explore overall survival (OS), transplant-specific outcomes, and reversal of the VEXAS clinical phenotype. We evaluated a total of 13 patients, all males and with a median age of 58 years (IQR, 52-63) at the time of transplant. The majority had concomitant MDS (n=10) while 2 presented with only autoinflammatory manifestations, and 1 had MPN. In line with the genomics of the disease, UBA1 mutations consisted of missense substitutions at the Met41 hotspot in 10 cases (n=6 Thr/3 Val/1 Leu), splice site (n=2 c.118-2A>C, c.118-1G>C), and the rare p.Ser56Phe at a median VAF of 73% (IQR, 45-84). All patients presented with anemia and had concomitant rheumatologic manifestations ( Fig1A) requiring specific treatment. VEXAS symptoms preceded the diagnosis of MDS/MPN in all but one case. Cytogenetics was normal in all VEXAS/MDS cases, which also displayed lower-risk IPSS-R scores with a median bone marrow blast of 2% (IQR, 1-4). Allo-HCT was performed at a median of 46 months (IQR, 22-59) from VEXAS onset. Before transplant, patients received a variety of both rheumatological (steroids/anti-interleukins) and hematological (azacitidine/ruxolitinib) treatments, with a high refractoriness (median number of lines 5, range 1-13). In all cases, stability of the clinical phenotype with no flares/uncontrolled manifestations was achieved at the time of the procedure. Conversely, in 3 MDS cases, clonal progression with acquisition of additional chromosomal alterations [+8, +21, t(12;16)] was evident. Overall, 62% of patients had a Karnofsky score >80% and low/intermediate (< 3) HCT-CI scores. Donor choice consisted of matched related in 23%, matched unrelated in 54%, mismatched related in 15%, and mismatched unrelated in 8% of the cohort. Peripheral blood was the preferred stem cell source in all but one case and conditioning regimen consisted of a variety of fludarabine-based strategies (n=4 MAC, n=9 RIC). GvHD prophylaxis was based on post-transplant cyclophosphamide (n=4), or serotherapy (n=8 ATG, n=1 alemtuzumab), in combination with cyclosporine and short-course methotrexate (n=5) or mycophenolate (n=4). All patients engrafted at a median time of 16 and 14 days for neutrophils and platelets, respectively. Overall, 92% of patients achieved full donor chimerism at last-follow-up. Grade II-IV acute GvHD occurred in 23% of cases, whereas only 1 patient presented with chronic extensive GvHD, which resolved 1-year after transplant. With a median time of 19 months (IQR, 8-42) from allo-HCT, 2-year OS was 82% ( Fig1B). Of note, 1 patient died because of infection and 1 because of CNS toxicity, whereas all cases with prolonged follow-up (>10 months) displayed resolution of the VEXAS clinical phenotypes and were able to discontinue immunosuppression between 2-8 months post-transplant. Allo-HCT remains the only curative option for MDS and is used to treat rheumatologic conditions refractory to conventional treatments. We present the largest series of allografted VEXAS cases. Given the hemato-inflammatory nature of the disease, allo-HCT may be a viable option in selected patients.

Automatic Generation of Alloreactivity-Reduced Donor Lymphocytes and Hematopoietic Stem Cells from the Same Mobilized Apheresis Product

CONCLUSION In vitro or in vivo depletion of alloreactive T cells can facilitate haplo-identical hematopoietic stem cell transplantation (HSCT). Very satisfactory transplant outcomes were thus reported for TCRαβ/CD19-depleted hematopoietic stem/progenitor cell (HSPC) grafts. However, the current semi-automatic manufacturing process, although robust, still requires a significant amount of time to be completed. Towards advancing and further facilitating large scale cell processing, a new TCRαβ/CD19 depletion module combined with a, previously described, CD45RA depletion module (to serve as allo-reactivity attenuated donor lymphocyte infusion) was established on the CliniMACS Prodigy. We evaluated six apheresis products from G-CSF-mobilized volunteer donors which were split, one portion each depleted of CD45RA + or of TCRα/β + and CD19 + cells. Products were assessed for recovery of HSPCs and other mature subsets, as well as depletion of targeted cells using flow cytometry. Effects of apheresis and product age post 48 hours storage at 2-8 °C as well as freeze-thawing on product viability and recovery of WBC and HPSCs were assessed by flow cytometry. Ten sequential depletions were technically uneventful, proceeding automatically with minimal hands-on time beyond tubing set installation. Depletion of CD45RA +, TCRα/β + and CD19 + cells was nearly complete, and at least equivalent to previous reports, achieving mean depletions of 4 log of targeted cells for both products. HSPC products retained TCRγ/δ + and NK cells. 48 hours storage of apheresis product was associated with the expected modest loss of HSPCs, but depletions proceeded efficiently. Depleted products were stable until at least 72 hours after apheresis with stem cell viabilities >90%. Freeze-thawing resulted in loss of NK cells; post-thaw recovery of viable CD45 + and HSPCs was >70% and in line with expectation. The closed, GMP-compatible process generates two separate medicinal products from the same mobilized apheresis product. The CD45RA-depleted products contained functional memory T cells, whereas the TCRαβ/CD19-depleted products included HSPCs, TCRγ/δ + and NK cells . Both products are predicted to be effectively depleted of GvH-reactivity while providing immunological surveillance, in support of haplo-identical HSCT.

STAG2 loss Induces HSC Programs By Modulating Accessibility of AP-1 Bound Enhancers

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are blood disorders characterized by clonal expansion of mutant hematopoietic stem and progenitor cells (HSPCs). Mutations in genes encoding the cohesin complex, including STAG2, RAD21, SMC3 and SMC1A, are frequent genetic drivers in MDS and AML. However, the mechanisms by which cohesin mutations lead to clonal expansion of HSPCs are not well understood. Cohesin is essential for sister chromatid cohesion, DNA damage repair, maintenance of chromatin architecture, and gene regulation. To investigate the impact of cohesin mutations on chromatin accessibility, we performed ATAC-Seq in a panel of six isogenic STAG2 wild type (WT) and knockout (KO) U937 leukemia cell lines. Among the total of 97,798 ATAC-Seq peaks, we identified significantly more STAG2 KO (8,859) versus WT (1,622) uniquely accessible chromatin sites, which were strongly enriched for intergenic regions. Integration of ChIP-Seq profiling of cohesin binding and histone modifications demonstrated cohesin binding and an increase in the active enhancer and promoter histone mark H3K27Ac in the STAG2 KO newly accessible chromatin, suggesting a direct role for cohesin in regulating the activity of such enhancers. Together, our data demonstrate that STAG2 KO-induced open chromatin may be enriched for novel STAG2 KO-specific enhancers. To further infer the regulatory function of these putative enhancers, we predicted binding of transcription factors (TFs) in ATAC-Seq-defined regions by performing TF footprinting using the Transcription factor Occupancy Prediction By Investigation of ATAC-seq Signal (TOBIAS) algorithm. We observed that binding of members of the AP-1 transcription family, including JUN FOS andATF, previously implicated in oncogenic transformation, was significantly enriched in the STAG2 KO cells (Figure 1A). Conversely, STAG2 KO cells lost CEBP TF binding in regions that lost chromatin accessibility. To identify potential regulatory targets of these TFs, we mapped TF footprints to annotated promoter regions and to enhancer-gene pairs determined by the Activity by Contact (ABC) model. We observed a strong correlation between predicted AP-1 binding and gene expression of putative regulated genes in STAG2 KO cells, further supporting the role of these newly accessible AP-1 bound sites as regulatory elements. To validate our findings using in vivo models of cohesin-mutant disease, we performed RNA-Seq and ATAC-Seq in HSPC isolated from a Tet2/Stag2 mouse model of MDS previously developed in our lab. We confirmed an increase in accessible chromatin and demonstrated a strong enrichment of predicted Ap-1 binding in intergenic regions, in agreement with our observations in the U937 model. Gene set enrichment analysis in the Tet2/Stag2-mutant versus WT HSPC revealed that putative targets of Ap-1 binding were enriched for hematopoietic stem cell gene signatures (Figure 1B) . This is consistent with our observation of clonal expansion of HSPCs in this model, and suggests a mechanism by which novel Ap-1 bound enhancers may facilitate expression of key genes involved in HSPC expansion and development of MDS/AML. To explore whether our findings were applicable to other published models of Stag2 mutant myeloid neoplasms, we performed TF footprinting using publicly available ATAC-Seq datasets generated in HSPC isolated from Stag2 and Runx1/Stag2 conditional knockout mice. We recapitulated increased chromatin accessibility and significant enrichment of cohesin-bound Ap-1 footprints in Stag2 mutant over WT conditions. Together, these data further validate conservation of STAG2 KO-induced AP-1 bound regulatory elements in human and mouse models of MDS and AML. In summary, we show that STAG2 loss leads to the establishment of a novel set of accessible intergenic regions with features of enhancer activity in vitro and in vivo. We identify multiple members of the AP-1 complex with enriched predicted binding at these putative enhancers, and demonstrate that AP-1 may drive expression of HSPC signatures in models of Stag2 mutant MDS in vivo. We illustrate here a potential role for the AP-1 complex in cohesin mutant myeloid malignancies, which we are currently functionally interrogating.

InCliniGene enables high-throughput andcomprehensive in vivo clonal tracking toward clinical genomics data integration.

High-throughput clonal tracking in patients under hematopoietic stem cell gene therapy with integrating vector is instrumental in assessing bio-safety and efficacy. Monitoring the fate of millions of transplanted clones and their progeny across differentiation and proliferation over time leverages the identification of the vector integration sites, used as surrogates of clonal identity. Although γ-tracking retroviral insertion sites (γ-TRIS) is the state-of-the-art algorithm for clonal identification, the computational drawbacks in the tracking algorithm, based on a combinatorial all-versus-all strategy, limit its use in clinical studies with several thousands of samples per patient. We developed the first clonal tracking graph database, InCliniGene (https://github.com/calabrialab/InCliniGene), that imports the output files of γ-TRIS and generates the graph of clones (nodes) connected by arches if two nodes share common genomic features as defined by the γ-TRIS rules. Embedding both clonal data and their connections in the graph, InCliniGene can track all clones longitudinally over samples through data queries that fully explore the graph. This approach resulted in being highly accurate and scalable. We validated InCliniGene using an in vitro dataset, specifically designed to mimic clinical cases, and tested the accuracy and precision. InCliniGene allows extensive use of γ-TRIS in large gene therapy clinical applications and naturally realizes the full data integration of molecular and genomics data, clinical and treatment measurements and genomic annotations. Further extensions of InCliniGene with data federation and with application programming interface will support data mining toward precision, personalized and predictive medicine in gene therapy. Database URL: https://github.com/calabrialab/InCliniGene.

Transfection of Vein Grafts with Early Growth Response Factor-1 Oligodeoxynucleotide Decoy: Effects on Stem-Cell Genes and Toll-like Receptor-Mediated Inflammation

The long-term patency of vein grafts is challenged by intimal hyperplasia. We sought to explore the intricate relationships between the transcription factor Egr-1, toll-like receptors (TLRs), and stem cell genes and also assessed oligodeoxynucleotide decoys (ODNs) as a strategy to prevent vein graft failures. A total of 42 New Zealand white rabbits were fed hyperlipidemic chow and classified into three groups. A double-stranded Egr-1 ODN was synthesized and infused in vein grafts prior to anastomosis with the common carotid artery. All vein grafts were retrieved at the conclusion of the predefined experimental period. Real-time quantitative polymerase chain reaction was performed to estimate expression patterns for several genes of interest. MYD88, TLR2-4, TLR8, NF-kB, TNF-α, IFNβ, and IFNγ; chemokines CCL4, CCL20, CCR2; numerous interleukins; and stem cell genes KFL4, NANOG, HOXA5, and HIF1α were universally downregulated in the ODN arm compared with the controls. By understanding these multifaceted interactions, our study offers actionable insights that may pave the way for innovative interventions in vascular reconstructions.

The Effect of Normal Follicular Fluid on the Differentiation of PCOS Ovarian Stem Cells into Oocyte-Like Cells.

Polycystic ovarian syndrome (PCOS) is one of the causes of infertility for which treatment methods do not have a high rate of pregnancy. In this study, the stem cells in the follicular fluid (FF) of patients were grown in the normal FF, and their differentiation into oocytes was evaluated. The FF of PCOS patients was centrifuged, and their cells were cultured with and without 20% normal FF for 2 weeks. The cells were evaluated for their morphology by inverted microscope and for markers of stem cells (NANOG and OCT4) and oocytes (zona pellucida (ZP) 2 and ZP3) by RT-PCR and immunocytochemistry. The amount of steroids was measured by enzyme-linked immunosorbent assay (ELISA). The cells were all round on day 0. After that, they had a heterogeneous morphology (fibroblast-like cells, epithelial-like cells, and round oocyte-like cells). In the first week, NANOG and OCT4 genes in the study group were less expressed than those in the control group (P < 0.0001) (~0.5-fold), while ZP2 and Z3 genes were more expressed (P < 0.0001) (~2-fold). In the second week, stem cell genes were more expressed in the control group (~2 fold), and oocyte genes were more expressed in the study group (P < 0.0001) (~2.5-3.11 fold). These results were also confirmed by immunocytochemistry. The amount of steroids was much higher in the study group (three times and five times in two weeks) (P < 0.0001). Stem cells can be obtained from the FF of PCOS, and normal FF has a positive effect on the growth and maturation of oocyte-like cells in vitro.

The RNA-binding protein CSDE1 promotes hematopoietic stem and progenitor cell generation via translational control of Wnt signaling.

In vertebrates, the earliest hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of specialized endothelial cells, hemogenic endothelial cells, in the aorta-gonad-mesonephros region through endothelial-to-hematopoietic transition. HSPC generation is efficiently and accurately regulated by a variety of factors and signals; however, the precise control of these signals remains incompletely understood. Post-transcriptional regulation is crucial for gene expression, as the transcripts are usually bound by RNA-binding proteins (RBPs) to regulate RNA metabolism. Here, we report that the RBP protein Csde1-mediated translational control is essential for HSPC generation during zebrafish early development. Genetic mutants and morphants demonstrated that depletion of csde1 impaired HSPC production in zebrafish embryos. Mechanistically, Csde1 regulates HSPC generation through modulating Wnt/β-catenin signaling activity. We demonstrate that Csde1 binds to ctnnb1 mRNAs (encoding β-catenin, an effector of Wnt signaling) and regulates translation but not stability of ctnnb1 mRNA, which further enhances β-catenin protein level and Wnt signal transduction activities. Together, we identify Csde1 as an important post-transcriptional regulator and provide new insights into how Wnt/β-catenin signaling is precisely regulated at the post-transcriptional level.

Gene and Cellular Therapies for Leukodystrophies.

Leukodystrophies are a heterogenous group of inherited, degenerative encephalopathies, that if left untreated, are often lethal at an early age. Although some of the leukodystrophies can be treated with allogeneic hematopoietic stem cell transplantation, not all patients have suitable donors, and new treatment strategies, such as gene therapy, are rapidly being developed. Recent developments in the field of gene therapy for severe combined immune deficiencies, Leber's amaurosis, epidermolysis bullosa, Duchenne's muscular dystrophy and spinal muscular atrophy, have paved the way for the treatment of leukodystrophies, revealing some of the pitfalls, but overall showing promising results. Gene therapy offers the possibility for overexpression of secretable enzymes that can be released and through uptake, allow cross-correction of affected cells. Here, we discuss some of the leukodystrophies that have demonstrated strong potential for gene therapy interventions, such as X-linked adrenoleukodystrophy (X-ALD), and metachromatic leukodystrophy (MLD), which have reached clinical application. We further discuss the advantages and disadvantages of ex vivo lentiviral hematopoietic stem cell gene therapy, an approach for targeting microglia-like cells or rendering cross-correction. In addition, we summarize ongoing developments in the field of in vivo administration of recombinant adeno-associated viral (rAAV) vectors, which can be used for direct targeting of affected cells, and other recently developed molecular technologies that may be applicable to treating leukodystrophies in the future.

What do osteoporosis and osteoarthritis have in common? An integrated study of overlapping differentially expressed genes in bone mesenchymal stem cells of osteoporosis and osteoarthritis

ObjectiveFor identification of aberrantly expressed genes in mesenchymal stem cells of osteoporosis (OP) and osteoarthritis (OA), Gene Expression Omnibus (GEO) datasets were integrated to investigate the intersection point. MethodsGSE35958 (osteoporosis) and GSE19664 (osteoarthritis) datasets were obtained from GEO database. The abnormally expressed genes were analyzed by GEO2R. Functional enrichment was explored by Metascape database and R software. The String database and Cytoscape software were used to build the protein–protein interaction network and identify hub genes. GSE35957 and GSE116925 were used as verification datasets. Single-cell analysis and pseudotime analysis were undertaken. CTDbase, Network Analyst, HPA database, HERB database and MIRW database were used to research the information, tissue and cell distribution, regulation, interaction and ingredients targeting the hub genes. Additionally, in vitro experiments such as RT-PCR, ALP staining and immunofluorescence were undertaken as verification tests. ResultsTen hub genes were identified in this study. All these genes play an important role in bone or cartilage generation. They have diagnostic values and therapeutic potential for OA and OP. Single-cell analysis visualized the cell distribution and pseudotime distribution of these genes. Some potential therapeutic ingredients of these genes were identified, such as curcumin, wogonin and glycerin. In vitro experiments, RT-PCR results showed that COL9A3 and MMP3 were downregulated and PTH1R was upregulated during osteogenic induction of BMSC. Immunohistochemical results showed the expression trend of MMP3 and COL2A1. ConclusionTen abnormal hub genes of osteoporosis and osteoarthritis were identified successfully by this study. They were important regulatory genes for healthy bone and cartilage. These genes could be the common connections between osteoporosis and osteoarthritis as well as treatment targets. Further study of the regulatory mechanism and treatment effects of these genes would be valuable. The results of this study could contribute to further research.