Stem Cell Regulatory Genes Research Articles

DPPA4 increases aggressiveness of pituitary neuroendocrine tumors by enhancing cell stemness.

Pituitary neuroendocrine tumors, PitNETs, are often aggressive and precipitate in distant metastases that are refractory to current therapies. However, the molecular mechanism in PitNETs' aggressiveness is not well understood. Developmental pluripotency-associated 4 (DPPA4) is known as a stem cell regulatory gene and overexpressed in certain cancers, but its function in the context of PitNETs' aggressiveness is not known. We employed both rat and human models of PitNETs. In the rat pituitary tumor model (RPT), we used prenatal-alcohol-exposed (PAE) female Fischer rats which developed aggressive PitNETs following estrogen treatment, while in the human pituitary tumor (HPT) model, we used aggressively proliferative cells from pituitary tumors of patients undergone surgery. Various molecular, cellular, and epigenetic techniques were used to determine the role of DPPA4 in PitNETs' aggressiveness. We show that DPPA4 is overexpressed in association with increased cell stemness factors in aggressive PitNETs of PAE rats and of human patients. Gene-editing experiments demonstrate that DPPA4 increases the expression of cell stemness and tumor aggressiveness genes and promotes proliferation, colonization, migration, and tumorigenic potential of PitNET cells. ChIP assays and receptor antagonism studies reveal that DPPA4 binds to canonical WINTs promoters and increases directly or indirectly the Wnt/β-catenin control of cell stemness, tumor growth, and aggressiveness of PitNETs. Epigenetic studies show involvement of histone methyltransferase in alcohol activation of DPPA4. These findings support a role of DPPA4 in tumor stemness and aggressiveness and provide a preclinical rationale for modulating this stemness regulator for the treatment of PitNETs.

Protective Function of Human Skin Mesenchymal Niches for Acute Myeloid Leukemia-Initiating Stem Cells

Leukemia cutis (LC) or leukemic cell infiltration in skin is one of the common extramedullary manifestations in acute myeloid leukemia (AML) and signifies a poorer prognosis. However, the pathogenesis and maintenance of LC have been understudied, limiting our understanding of its impact on AML progression and relapse. We recently have shown that the AML cells infiltrated in the mouse skin during steady state and after chemotherapy were capable to regenerate AML after serial transplantation (Sandhow et al., J. Exp. Med, 2023). Further characterization of the skin mesenchymal stem cells (MSCs) suggests that skin MSCs could maintain AML-initiating stem cells (LSCs) and protect them from chemotherapy, even to a greater degree than their bone marrow (BM) counterparts. These data suggest the protective role of skin mesenchymal niche in AML LSCs during chemotherapy. However, it remains unexplored whether human skin MSCs exert the same functional impact on human AML cells. We here have explored the role of human skin MSCs in AML maintenance and survival during chemotherapy by RNA sequencing and in vitro co-culture of patient-derived AML cells with primary human skin MSCs in comparison with BM MSCs. We first by prospective characterization identified human skin MSCs with a phenotype of CD45-CD235a-CD31-CD44+CD146- from healthy donors. RNA sequencing and Q-PCR revealed expression of key hematopoietic stem cell (HSC) regulatory genes including THPO, SCF, CXCL12 and LAMA4 in human skin MSCs. Interestingly, while HSC maintenance genes such as THPO and KITLG were significantly upregulated in skin MSCs (p<0.0001 and p<0.05, respectively), HSC negative regulator SPP1 was downregulated (p=0.05), pointing to a possible better hematopoiesis-supportive function of skin MSCs. In line with this, our functional studies indicated that skin MSCs displayed a superior protective effect on human AML cell line THP-1 than their BM cell counterparts during cytarabine (Ara-C) treatment in the in vitro co-culture. There were more residual chemo-resistant AML cells, reflected in the higher number of CD36+ AML cells after treatment with Ara-C in cocultures with skin MSCs, compared to that with BM MSCs (p=0.0065). This result was confirmed by functional assays showing the increased number of cobblestone area-forming cells (p=0.0298) and colony-forming units (p=0.038) within the residual AML cells co-cultured with skin MSCs. To investigate the mechanisms involved in the protective function of human skin MSCs, we have performed RNA-sequencing of the human skin and BM MSCs before and after co-culture with AML cells. This has revealed potential unique molecular pathways between AML and skin MSCs. Furthermore, there were dramatically less altered genes in the skin MSCs compared to BM MSCs after 3 day-coculture with THP1 AML cells (221 vs 1761 altered genes with adjusted p values <0.05). These data indicate that skin MSCs are likely more resistant to AML-remodeling, which could in turn maintain AML cells in quiescence status, thereby protecting AML cells from Ara-C treatment. This speculation is currently under validation. In addition, gene set enrichment analysis showed upregulated genes associated with fatty acid metabolisms and ribosome in skin MSCs compared to BM MSCs, providing molecular evidence for a potential better metabolic support of skin MSCs for AML cells. Taken together, our preliminary data indicate an important role of human skin MSCs in supporting and protecting AML cells during Ara-C treatment. We are further investigating the underlying mechanisms.

HOX genes in stem cells: Maintaining cellular identity and regulation of differentiation.

Stem cells display a unique cell type within the body that has the capacity to self-renew and differentiate into specialized cell types. Compared to pluripotent stem cells, adult stem cells (ASC) such as mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) exhibit restricted differentiation capabilities that are limited to cell types typically found in the tissue of origin, which implicates that there must be a certain code or priming determined by the tissue of origin. HOX genes, a subset of homeobox genes encoding transcription factors that are generally repressed in undifferentiated pluripotent stem cells, emerged here as master regulators of cell identity and cell fate during embryogenesis, and in maintaining this positional identity throughout life as well as specifying various regional properties of respective tissues. Concurrently, intricate molecular circuits regulated by diverse stem cell-typical signaling pathways, balance stem cell maintenance, proliferation and differentiation. However, it still needs to be unraveled how stem cell-related signaling pathways establish and regulate ASC-specific HOX expression pattern with different temporal-spatial topography, known as the HOX code. This comprehensive review therefore summarizes the current knowledge of specific ASC-related HOX expression patterns and how these were integrated into stem cell-related signaling pathways. Understanding the mechanism of HOX gene regulation in stem cells may provide new ways to manipulate stem cell fate and function leading to improved and new approaches in the field of regenerative medicine.

Prediction of intestinal stem cell regulatory genes from Drosophila gut damage model created using multiple inducers: Differential gene expression-based protein-protein interaction network analysis

Intestinal tissue functions in innate immunity to prevent the entry of harmful substances, and to maintain homeostasis through the constant proliferation of intestinal stem cells (ISC). To understand the mechanisms which regulate ISC in response to gut damage, we identified 81 differentially expressed genes (DEGs) through RNA-seq analysis after oral administration of three intestinal-damaging substances to Drosophila melanogaster. Through protein-protein interaction (PPI) and functional annotation studies, the top 22 DEGs ordered by the number of nodes in the PPI network were analyzed in relation to cell development. Through network topology analysis, we identified 12 essential seed genes. From this we confirmed that p53, RpL17, Fmr1, Stat92E, CG31343, Cnot4, CG9281, CG8184, Evi5, and to were essential for ISC proliferation during gut damage using knockdown RNAi Drosophila. This study presents a method for identifying candidate genes relating to intestinal damage that has scope for furthering our understanding of gut disease.

Relevance of renal cell carcinoma angiogenesis and tumor stem cells

Objective: To investigate the relationship between tumor angiogenesis and renal cancer stem cells. Methods: The primary cell culture method was used to extract and isolate RCSCs, and then qRT-PCR and Western blot methods were used to detect the expression levels of the kidney cancer stem cell markers CD105 and Sox2 genes and proteins from different MVD kidney cancer tissues. Using TCGA database, analyze the correlation between tumor angiogenesis markers and tumor stem cell regulatory genes. Results: The stem cell markers CD105 and Sox2 genes in RCSCs derived from high MVD kidney cancer tissues were respectively increased by (2.34±1.77) times and (3.92±1.41) times (PCD105<0.01, PSox2<0.05)and protein levels were increased by (5.12±3.31) times and (4.90±3.30) times(PCD105<0.05, PSox2<0.01).Meanwhile,up to 30% of stem cell promoting stemness regulatory genes are positively correlated with angiogenesis genes CD31/PECAM1 and KDR, and 64 genes are also strongly positively correlated with CD31/PECAM1 and KDR genes. Conclusion: The high microvessel density of kidney cancer is strongly correlated with the existence of renal carcinoma stem cells.

Empty spiracles homeobox genes EMX1 and EMX2 regulate WNT pathway activation in sarcomagenesis

BackgroundSarcomas are a very heterogeneous group of tumors with intrinsic developmental programs derived from the cell of origin. This implies a functional hierarchy inside tumors governed by sarcoma stem cells. Therefore, genetic and/or epigenetic changes profoundly affect the biology of sarcoma tumor stem cells. EMX genes are proposed to be transcription factors that are involved in the sarcomagenesis process, regardless of the neural or mesodermal embryological sarcoma origin. It has been shown that EMX1 or EMX2 overexpression reduces tumorigenic properties, while reducing the levels of these genes enhances these properties. Furthermore, it has been shown that EMX genes decrease the expression of stem cell regulatory genes and the stem cell phenotype. Taken together, these results indicate that the EMX1 and EMX2 genes negatively regulate these tumor-remodeling populations or sarcoma stem cells, acting as tumor suppressors in sarcoma.MethodsBioinformatic analysis, quantitative mRNA and protein expression analysis, cell models of sarcoma by ectopic expression of EMX genes. By cell biology methods we measured tumorigenesis and populations enriched on stem cell phenotypes, either in vitro or in vivo.ResultsIn this work, we showed that the canonical Wnt pathway is one of the mechanisms that explains the relationships of EMX1/EMX2 and stem cell genes in sarcoma. The Wnt-EMX1/EMX2 relationship was validated in silico with sarcoma patient datasets, in vitro in primary derived sarcoma cell lines, and in vivo. EMX expression was found to negatively regulate the Wnt pathway. In addition, the constitutive activation of the Wnt pathway revers to a more aggressive phenotype with stem cell properties, and stemness gene transcription increased even in the presence of EMX1 and/or EMX2 overexpression, establishing the relationship among the Wnt pathway, stem cell genes and the EMX transcription factors.ConclusionsOur data showed that Empty Spiracles Homeobox Genes EMX1 and EMX2 represses WNT signalling and activation of WNT pathway bypass EMX-dependent stemness repression and induces sarcomagenesis. These results also suggest the relevance of the Wnt/b-catenin/stemness axis as a therapeutic target in sarcoma.

Regulation of sarcomagenesis by the empty spiracles homeobox genes EMX1 and EMX2

The EMX (Empty Spiracles Homeobox) genes EMX1 and EMX2 are two homeodomain gene members of the EMX family of transcription factors involved in the regulation of various biological processes, such as cell proliferation, migration, and differentiation, during brain development and neural crest migration. They play a role in the specification of positional identity, the proliferation of neural stem cells, and the differentiation of certain neuronal cell phenotypes. In general, they act as transcription factors in early embryogenesis and neuroembryogenesis from metazoans to higher vertebrates. The EMX1 and EMX2’s potential as tumor suppressor genes has been suggested in some cancers. Our work showed that EMX1/EMX2 act as tumor suppressors in sarcomas by repressing the activity of stem cell regulatory genes (OCT4, SOX2, KLF4, MYC, NANOG, NES, and PROM1). EMX protein downregulation, therefore, induced the malignance and stemness of cells both in vitro and in vivo. In murine knockout (KO) models lacking Emx genes, 3MC-induced sarcomas were more aggressive and infiltrative, had a greater capacity for tumor self-renewal, and had higher stem cell gene expression and nestin expression than those in wild-type models. These results showing that EMX genes acted as stemness regulators were reproduced in different subtypes of sarcoma. Therefore, it is possible that the EMX genes could have a generalized behavior regulating proliferation of neural crest-derived progenitors. Together, these results indicate that the EMX1 and EMX2 genes negatively regulate these tumor-altering populations or cancer stem cells, acting as tumor suppressors in sarcoma.

The response of PIK3CA/KRAS-mutant colorectal cancer stem-like cells to RGD-peptide FraC produced by the strawberry anemone: A promising water-soluble peptide-based inhibitor of metastasis-driver gene CXCR4, stem cell regulatory genes and self-renewal

Colorectal cancer (CRC) is a stem cell-based disease. PIK3CA/KRAS-mutant CRC stem cells (CRCSCs) display high self-renewal, metastatic properties, high activity of PI3K and KRAS signaling pathways with chemoresistant phenotypes. Recently, RGD peptide (containing Arg-Gly-Asp motif)-based therapy of solid tumor cells has attracted much attention. However, little is known whether this method can target self-renewal capacity, key effectors of PI3K and KRAS signaling pathways such as metastasis-driver gene CXCR4 and stem cell regulatory genes with caspase-3 reactivation in CRCSCs overexpressing RGD-dependent integrins.The sea anemone Actinia fragacea produces a water-soluble RGD-peptide fragacea toxin C (FraC) suggesting the possible activity of FraC against PIK3CA/KRAS-mutant CRCSCs.Recombinant FraC was expressed via pET-28a(+)-FraC in E. coli and purified through affinity chromatography followed by performing SDS-PAGE and hemolytic activity assay. Next, PIK3CA/KRAS-mutant HCT-116 cells that serve as an attractive model for CRCSCs were treated with FraC. Thereafter, cell numbers, viability, proliferation, LDH activity, cytotoxicity index, CXCR4 and pluripotency network genes expression, self-renewal capacity, caspase-3 activity with apoptosis were evaluated. Caspase-1, -2, -3,…, -9 sequences were analyzed for RGD-binding motifs. FraC sequence and structure were also evaluated by bioinformatics software.FraC altered cellular morphology to round shapes and disrupted cell connections. 48 h post-treatment with 0.056˗ to 7.2 μM FraC resulted in 12 %–99 % and 8 %–97.6 % decreases in cell numbers and viabilities respectively and increased LDH activity by 0.2 %–66.7 % in a dose-dependent manner. The results of the cytotoxicity index showed that FraC induces significant toxicity on HCT-116 cells compared to PBMCs and Huvec cells. FraC dramatically decreased the expression of CXCR4 and pluripotency network genes Bmi-1, Sox-2, Oct-4 and Nanog followed by remarkable decreases in self-renewal capacity ranged from 91- to 0 colonies per well for 0.056˗ to 3.6 μM FraC after 2 weeks. Caspase-3 was found to contain an RGD-binding motif and its activity increased with increasing FraC concentrations followed by apoptosis induction. Potential RGD-binding motifs for FraC were also found in caspase-1, -7, -8 and -9.Unique advantages of FraC peptide, such as low molecular weight, water solubility, high sensitivity of CRC stem-like cells with more selective toxicity to this compound, targeting tumor cell membrane and self-renewal capacity along with the modulation of CXCR4 and stem cell regulatory genes as upstream and downstream effectors of undruggable PI3K and KRAS signaling pathways may open up avenues for FraC peptide-based therapy of PIK3CA/KRAS-mutant CRCSCs with lower toxicity on healthy cells.

MiR-376c-3p modulates the properties of breast cancer stem cells by targeting RAB2A.

MicroRNAs (miRNAs/miRs) negatively regulate gene expression and participate in various cellular processes. miRNA dysregulation is associated with cancer progression. The present study aimed to identify the miRNAs that participate in breast cancer tumorigenesis and determine the mechanism that underlies this. miRNA microarray data analysis and validation assays indicated that miR-376c-3p was downregulated in breast tumour tissues and breast cancer stem cells (BCSCs) compared with adjacent non-cancerous tissues and MCF-10A cells, respectively. Ras-related protein Rab-2A (RAB2A) was predicted as a target of miR-376c-3p, which was confirmed by conducting further experiments. miR-376c-3p regulated the BCSC population and the expression of stem cell regulatory genes by targeting RAB2A. By performing mammosphere, Cell Counting Kit-8, colony formation and transwell invasion assays, it was demonstrated that miR-376c-3p also inhibited BCSC self-renewal, proliferation and invasion by regulating RAB2A expression. Using a xenograft mouse model, it was revealed that miR-376c-3p overexpression suppressed breast cancer growth in vivo. In conclusion, the results indicated that miR-376c-3p targeted RAB2A to regulate BCSC fate and properties; therefore, miR-376c-3p may serve as a potential therapeutic target for breast cancer.

Small ribonucleoprotein particle protein SmD3 governs the homeostasis of germline stem cells and the crosstalk between the spliceosome and ribosome signals in Drosophila.

The ribonucleoprotein (RNP) spliceosome machinery triggers the precursor RNA splicing process in eukaryotes. Major spliceosome defects are implicated in male infertility; however, the underlying mechanistic links between the spliceosome and the ribosome in Drosophila testes remains largely unresolved. Small ribonucleoprotein particle protein SmD3 (SmD3) is a novel germline stem cell (GSC) regulatory gene identified in our previous screen of Drosophila testes. In the present study, using genetic manipulation in a Drosophila model, we demonstrated that SmD3 is required for the GSC niche and controls the self-renewal and differentiation of GSCs in the testis. Using in vitro assays in Schneider 2 cells, we showed that SmD3 also regulates the homeostasis of proliferation and apoptosis in Drosophila. Furthermore, using liquid chromatography-tandem mass spectrometry methods, SmD3 was identified as binding with ribosomal protein (Rp)L18, which is a key regulator of the large subunit in the ribosome. Moreover, SmD3 was observed to regulate spliceosome and ribosome subunit expression levels and controlled spliceosome and ribosome function via RpL18. Significantly, our findings revealed the genetic causes and molecular mechanisms underlying the stem cell niche and the crosstalk between the spliceosome and the ribosome.-Yu, J., Luan, X., Yan, Y., Qiao, C., Liu, Y., Zhao, D., Xie, B., Zheng, Q., Wang, M., Chen, W., Shen, C., He, Z., Hu, X., Huang, X., Li, H., Chen, B., Zheng, B., Chen, X., Fang, J. Small ribonucleoprotein particle protein SmD3 governs the homeostasis of germline stem cells and the crosstalk between the spliceosome and ribosome signals in Drosophila.

Latexin Inhibition Mitigates Irradiation Induced Hematopoietic Injury

Radiation-associated bone marrow (BM) injury is one of the most serious limiting factors of radiotherapy. Radiation-induced hematopoietic injury, no matter how transient or long lasting, can ultimately impair HSC function and decrease the HSC reserve, leading to increased risk for the development of BM failure or cancer. However, molecular mechanisms underlying radiation-induced HSC functional decline are largely unknown.We previously identified a stem cell regulatory gene, latexin (Lxn), as a novel negative regulator of HSCs in mice. HSCs in Lxn knockout mice (Lxn-/-) had increased self-renewal and survival. In our new findings, we surprisingly found that Lxn-/- mice had the significant survival advantages under lethal dose of total body irradiation (TBI). We further found that HSCs and hematopoietic progenitor cells (HPCs), measured by immunophenotypes and colony assay, recovered much faster in Lxn-/- mice than wild-type mice (WT) within one month after sub-lethal dose of TBI. The better preserved HSC/HPC pool was due to the decreased apoptosis in which the percentage of Annexin V + PI- apoptotic HSCs/HPCs cells was significantly lower in Lxn-/- mice than WT mice. These data suggest that Lxn inactivation protects HSCs and HPCs from radiation-induced cell death, thus mitigating acute hematopoietic suppression and conferring a survival advantage.To determine the long-term effect of TBI on Lxn-/- HSCs, we performed limiting dilution competitive repopulation unit assay (CRU), and found that Lxn-/- CRU was significantly higher than WT CRU. Moreover, we performed serial transplantation experiment, and found that Lxn-/- HSC continuously regenerated blood and bone marrow cells even at the 4th round of transplantation whereas WT HSCs were exhausted. These data provide robust evidence that Lxn inactivation protects functional long-term HSCs from radiation-induced injury. Radiation can increase the risk of hematological malignancy later in the life. We thus maintained a group of mice that were subject to either a single dose of 6.5Gy TBI or split low doses of TBI (2 Gy daily for 6 days), and monitored their gross condition and blood cell counts for 20 months. At 20 month post-radiation, we performed bone marrow analysis and histopathology analysis. We found that Lxn-/- mice did not spontaneously develop hematopoietic malignancies, their bone marrow HSCs/HPCs had normal population size, and bone marrow had normal histopathology. These data suggest that Lxn inactivation mitigates radiation-induced short-term myelosuppression and long-term HSC functional impairment without induction of hematologic malignancy.At the molecular level, we previously reported that Lxn sensitized leukemogenic cells to gamma-irradiation-induced cell-cycle arrest and cell death through Rps3 pathway, and Rps3 was a binding protein of Lxn. Rps3 has been shown to be involved in the NFkB pathway. We found that Rps3 bound Lxn in primary hematopoietic stem and progenitor cells (HSPCs) using Co-IP assay. Lxn-/- HSPCs had the increased expression of Rps3 and NFkB p65 before or post-irradiation. Knockdown of Rps3 in Lxn-/- HSPCs decreased NFkB p65 and increased radiation-induced apoptosis. Moreover, when Lxn-/- HSPCs were treated with NFkB p65 specific inhibitor, the similar phenotypes were also shown, suggesting that Lxn functions through Rps3-NFkB-mediated pro-survival pathway in primary HSPCs. We are currently proving this molecular pathway using the in vivo model by crossing p65 knockout mice with Lxn-/- mice.In conclusion, latexin inhibition mitigates irradiation induced hematopoietic injury via Rps3-NFkB-mediated pro-survival pathway. DisclosuresNo relevant conflicts of interest to declare.

Plant stem cells: what we know and what is anticipated.

Plant stem cell research is of interest due to stem cells ability of unlimited division, therapeutic potential and steady supply to provide precursor cells. Their isolation and culture provides the important source for the production of homogenous lines of active constituents that allow large-scale production of various metabolites. The process of dedifferentiation and reversal to pluripotent cells involves the various pathways genes related to the stem cells and are associated to each other for maintaining a specific niche. Domains such as niche dynamics and maintenance signaling can be used for the identification of genes for stem cell niche. Significant findingshave beenachieved in the past on plant stem cells however our understanding towards mechanisms underlying some specific phenomenon like dedifferentiation, regulation, niche dynamics is still in infancy. The present review is based on the past research efforts and also pave a way forward for the future anticipation in the field of development of cell cultures for the production of active metabolites on large scale andundertanding transcriptional regulation of stem cell genes involved in niche signaling.

Theranostic Niosomes for Efficient siRNA/MicroRNA Delivery and Activatable Near-Infrared Fluorescent Tracking of Stem Cells.

RNA interference-mediated gene regulation in stem cells offers great potential in regenerative medicine. In this study, we developed a theranostic platform for efficient delivery of small RNAs [small interfering RNA (siRNA)/microRNA (miRNA)] to human mesenchymal stem cells (hMSCs) to promote differentiation, and meanwhile, to specifically label the transfected cells for the in vivo tracking purpose. We encapsulated indocyanine green (ICG) in a nonionic surfactant vesicle, termed "niosome", that is mainly composed of a nonionic surfactant sorbitan monooleate (Span 80) and a cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). This novel ICG-containing niosome system (iSPN) demonstrated highly efficient siRNA and miRNA delivery in hMSCs. Specific inhibition of miR-138, a negative regulator of osteoblast differentiation, was achieved by iSPN/miR-138, which significantly promoted osteogenesis of hMSCs. Furthermore, iSPN exhibited OFF/ON activatable fluorescence upon cellular internalization, resulting in efficient near-infrared labeling and the capability to dynamically monitor stem cells in mice. In addition, iSPN/siRNA achieved simultaneous long-term cell tracking and in vivo gene silencing after implantation in mice. These results indicate that our theranostic niosomes could represent a promising platform for future development of stem cell-based therapy.

Role of heterotrimeric Gα proteins in maize development and enhancement of agronomic traits.

Plant shoot systems derive from the shoot apical meristems (SAMs), pools of stems cells that are regulated by a feedback between the WUSCHEL (WUS) homeobox protein and CLAVATA (CLV) peptides and receptors. The maize heterotrimeric G protein α subunit COMPACT PLANT2 (CT2) functions with CLV receptors to regulate meristem development. In addition to the sole canonical Gα CT2, maize also contains three eXtra Large GTP-binding proteins (XLGs), which have a domain with homology to Gα as well as additional domains. By either forcing CT2 to be constitutively active, or by depleting XLGs using CRISPR-Cas9, here we show that both CT2 and XLGs play important roles in maize meristem regulation, and their manipulation improved agronomic traits. For example, we show that expression of a constitutively active CT2 resulted in higher spikelet density and kernel row number, larger ear inflorescence meristems (IMs) and more upright leaves, all beneficial traits selected during maize improvement. Our findings suggest that both the canonical Gα, CT2 and the non-canonical XLGs play important roles in maize meristem regulation and further demonstrate that weak alleles of plant stem cell regulatory genes have the capacity to improve agronomic traits.

Latexin Inactivation Enhances Survival and Long-Term Engraftment ofHematopoietic Stem Cells and Expands the Entire Hematopoietic System in Mice.

SummaryNatural genetic diversity offers an important yet largely untapped resource to decipher the molecular mechanisms regulating hematopoietic stem cell (HSC) function. Latexin (Lxn) is a negative stem cell regulatory gene identified on the basis of genetic diversity. By using an Lxn knockout mouse model, we found that Lxn inactivation in vivo led to the physiological expansion of the entire hematopoietic hierarchy. Loss of Lxn enhanced the competitive repopulation capacity and survival of HSCs in a cell-intrinsic manner. Gene profiling of Lxn-null HSCs showed altered expression of genes enriched in cell-matrix and cell-cell interactions. Thrombospondin 1 (Thbs1) was a potential downstream target with a dramatic downregulation in Lxn-null HSCs. Enforced expression of Thbs1 restored the Lxn inactivation-mediated HSC phenotypes. This study reveals that Lxn plays an important role in the maintenance of homeostatic hematopoiesis, and it may lead to development of safe and effective approaches to manipulate HSCs for clinical benefit.

ZEB1 regulates glioma stemness through LIF repression

The identification of a stem cell regulatory gene which is aberrantly expressed in glioma and associated with patient survival would increase the understanding of the role of glioma cancer stem cells (GCSCs) in the virulence of gliomas. Interrogating the genomes of over 4000 brain cancers we identified ZEB1 deletion in ~15% (grade II and III) and 50% of glioblastomas. Meta-analysis of ZEB1 copy number status in 2,988 cases of glioma revealed disruptive ZEB1 deletions associated with decreased survival. We identified ZEB1 binding sites within the LIF (stemness factor) promoter region, and demonstrate LIF repression by ZEB1. ZEB1 knockdown in GCSCs caused LIF induction commensurate with GCSC self-renewal and inhibition of differentiation. IFN-γ treatment to GCSCs induced ZEB1 expression, attenuating LIF activities. These findings implicate ZEB1 as a stem cell regulator in glioma which when deleted leads to increased stemness, tumorigenicity and shortened patient survival.

RNAi and MicroRNA-Mediated Gene Regulation in Stem Cells.

Recently, RNAi and microRNAs (miRNAs) have become important tools to investigate the regulatory mechanism of stem cell maintenance and differentiation. In this short review, we give a brief overview of the discovery history, functions, and mechanisms of RNAi and miRNAs. We also discuss the RNAi as a tool to study the stem cell function and the potential future practical applications.

Identification and validation strategy for hematopoietic stem cell regulatory genes

In this study we used large data sets of genomic γ-retroviral (γRV) integration sites (IS) from a clinical gene therapy trial to identify HSC regulatory genes and developed a pooled competitive overexpression strategy for functional validation in vivo. We hypothesized that common integration sites (CIS) indicate actively transcribed genes in human HSCs or genetic loci, which are deregulated by viral enhancer and promotor elements nearby transcription start (TSS) sites, leading to clonal expansion. Systematic analysis of the IS repertoire of 10 Wiskott-Aldrich syndrome patients revealed 130,699 unique IS in the vicinity of 12,437 genes. Applying stringent selection criteria based on the cluster size and distance of IS to TSS, we identified 1,545 gene associated CIS. Approximately 25% of the top ranked CIS are located in the vicinity of published HSC regulators, such as MECOM, LMO2 or HMGA2. Moreover, pathway analysis using the CIS tagged gene signature revealed significant enrichment for hematological system development and function as well as immune response pathways, thus strongly validating our selection strategy. To study the influence of all 20 top ranked candidate genes in parallel, we generated an inducible and barcoded lentiviral overexpression library, consisting of 2 unique barcodes (BCs) per gene and 10 barcoded GFP-only controls. Nested amplification of BCs and indexing of 96 samples/lane (HiSeq 2500) was established and tested intensively in a “one BC/sample” experiment, showing averagely 1.37E6 reads per sample with only 11 off target reads per BC (0.0008%±2.8E-6 [SEM]). Pooled transduction of LSKRosa26-rtTA cells and Doxycycline administration 4 weeks after transplantation showed long-term GFP expression in 1° and 2° recipients (94.4%±0.4 engraftment; 4.2%±1.1 GFP+ [SEM]). Serial bleedings and multiplexed HT-sequencing of amplified BCs from gDNA of B-, T-, and myeloid cells revealed a complete representation of all BCs in all mice (n=23) before and after Dox administration with 15,643±656 (SEM) read counts/BC at week 4 and 46,222±3276 (SEM) read counts/BC at week 16. Our pooled competitive overexpression strategy allows us to study 20 genes in parallel facilitating the discovery of novel key players in hematopoiesis.

RNA Splicing Modulation Impairs Acute Myeloid Leukemia Stem Cell Maintenance

IntroductionDisease relapse is the leading cause of death in secondary AML (sAML), which evolves from antecedent hematologic disorders like myelodysplastic syndrome (MDS) or myeloproliferative neoplasms (MPNs) or following exposure to chemotherapy. Persistence of therapy-resistant leukemia stem cells (LSC) harboring enhanced survival and self-renewal capacity has been linked to high relapse rates in sAML. Previously, we showed that missplicing of a stem cell regulatory gene, GSK3 b, and splice isoform switching favoring pro-survival BCL2 family isoform expression promoted generation of therapy-resistant LSC (Abrahamsson et al PNAS 2009; Goff et al Cell Stem Cell 2013). However, whether aberrant pre-mRNA splicing promotes sAML LSC generation, in the absence of mutation, and if pharmacological splicing modulation impairs LSC maintenance, in a mutation-independent manner, has not been elucidated.Methods and ResultsComparative RNA-sequencing and gene set enrichment analyses revealed significant alterations in splicing factor gene expression in purified progenitors from untreated sAML compared with normal samples. In addition, using an isoform-specific alignment algorithm, we established a sAML LSC splice isoform expression signature that identified increased expression of select transcripts, e.g. CD82 and PTK2B. Thus, we investigated the LSC inhibitory efficacy of a stable, potent splicing modulatory agent, 17S -FD-895, in humanized AML LSC stromal co-culture and primagraft assays. Notably, there was a dose-dependent reduction in AML LSC (n=4) survival and self-renewal after in vitro 17S -FD-895 treatment, with a favorable therapeutic index compared to normal controls (n=3, p<0.01). Splicing reporter activity and PCR analyses revealed rapid and potent 17S -FD-895-induced alterations in splicing, promoting pro-apoptotic isoform expression and intron inclusion in the stem cell regulatory gene MCL1. Also, 17S -FD-895 restored normal expression patterns of PTK2B, and MCL1-L/S and BLC2-L/S expression ratios.Flow cytometric analyses in AML LSC primagraft models treated with 17S -FD-895 (5-10 mg/kg delivered intravenously in 3 doses over 2 weeks) revealed a decrease in human stem (CD45+ CD34+ CD38- Lin-, 68% reduction in the spleens of the 10 mg/kg group versus vehicle controls, n=5 mice per group, p<0.05) and progenitor (CD45+ CD34+ CD38+ Lin-, 80% reduction to nearly zero in the spleens of the 10 mg/kg group versus vehicle controls, p=0.08) cell frequencies. Furthermore, MCL1-L/S and BCL2-L/S expression ratios were significantly reduced in LSC-enriched fractions from 17S -FD-895-treated mice compared to vehicle controls. Consistent with a reduction in functional LSC burden after 17S -FD-895 treatment, subsequent serial transplantation studies showed a 47-65% reduction in leukemic burden in the hematopoietic tissues of recipients of CD34+ cells from mice in the 10 mg/kg treatment group versus vehicle controls (n=5 mice per group, p<0.05).ConclusionsHere we demonstrate that a potent and stable splicing modulatory agent, 17S -FD-895, normalized sAML-specific splice isoform expression patterns as well as MCL1-L/S and BLC2-L/S ratios. Moreover, pharmacologic splicing modulation reduced AML LSC survival and self-renewal in a dose-dependent manner in both in vitro and in vivo models with a favorable therapeutic index. Further evaluation of this compound as a splicing-targeted single agent or combined with standard of care therapy may reduce or eradicate LSC burden in therapy-resistant sAML. In addition, LSC-specific splice isoforms may represent important biomarkers that could be developed as companion diagnostics for splicing-targeted therapies in sAML and other recalcitrant malignancies. DisclosuresJamieson:Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.

The Drosophila bag of marbles Gene Interacts Genetically with Wolbachia and Shows Female-Specific Effects of Divergence.

Many reproductive proteins from diverse taxa evolve rapidly and adaptively. These proteins are typically involved in late stages of reproduction such as sperm development and fertilization, and are more often functional in males than females. Surprisingly, many germline stem cell (GSC) regulatory genes, which are essential for the earliest stages of reproduction, also evolve adaptively in Drosophila. One example is the bag of marbles (bam) gene, which is required for GSC differentiation and germline cyst development in females and for regulating mitotic divisions and entry to spermatocyte differentiation in males. Here we show that the extensive divergence of bam between Drosophila melanogaster and D. simulans affects bam function in females but has no apparent effect in males. We further find that infection with Wolbachia pipientis, an endosymbiotic bacterium that can affect host reproduction through various mechanisms, partially suppresses female sterility caused by bam mutations in D. melanogaster and interacts differentially with bam orthologs from D. melanogaster and D. simulans. We propose that the adaptive evolution of bam has been driven at least in part by the long-term interactions between Drosophila species and Wolbachia. More generally, we suggest that microbial infections of the germline may explain the unexpected pattern of evolution of several GSC regulatory genes.