Stem Cell Origin Research Articles

Stem Cells in Cardiovascular Medicine: Historical Overview and Future Prospects.

Cardiovascular diseases remain the leading cause of death in the developed world, accounting for more than 30% of all deaths. In a large proportion of these patients, acute myocardial infarction is usually the first manifestation, which might further progress to heart failure. In addition, the human heart displays a low regenerative capacity, leading to a loss of cardiomyocytes and persistent tissue scaring, which entails a morbid pathologic sequela. Novel therapeutic approaches are urgently needed. Stem cells, such as induced pluripotent stem cells or embryonic stem cells, exhibit great potential for cell-replacement therapy and an excellent tool for disease modeling, as well as pharmaceutical screening of novel drugs and their cardiac side effects. This review article covers not only the origin of stem cells but tries to summarize their translational potential, as well as potential risks and clinical translation.

Reconstruction of Human AML Reveals Stem Cell Origin and Therapeutic Targets for Treatment Resistant CD34-/Lo MLL-Rearranged Leukemia

Identification of the origins of acute myeloid leukemia (AML) stem cells has been a holy grail for a better understanding of the developmental biology of the disease and the design of effective treatments. Studies using primary AML samples on patient-derived xenograft models identified AML stem cells in multiple different CD34/CD38 cellular fractions, which shared similar immunophenotypes of hematopoietic stem/progenitor cells including hematopoietic stem cells (HSCs), lymphoid-primed multipotent progenitors (LMPPs) and granulocyte-macrophage progenitors (GMPs). However inference of cells-of-origin based on the retrospective approach has major limitations as the reported HSC/LMPP/GMP-like AML stem cells are phenotypically different from their normal counterparts; and AML stem cells identified in late developmental stages do not necessarily maintain the same immunophenotypes of the disease initiating (pre-leukemic) cells, which can retain a relatively normal differentiation potential, and only their descendants acquire additional events becoming AML stem cells. While prospective disease modelling using mouse cells has provided unique insights into the potential origins of AML stem cells, human and mouse cells have different transformation requirements, distinct telomere biology, and a significant degree divergence of transcriptional regulation, chromatin state and gene regulatory networks that can profoundly affect their transformation potential and associated cancer biology. Therefore we reason that deconstruction of AML stem cell hierarchy from primary human samples followed by reconstruction of the corresponding human disease using candidate cell populations will give novel insights into this issue. Given that AML is a highly heterogeneous disease, our study initially focused on genetically well-defined MLL-rearranged AML frequently found in both infant and adult leukemia. By analyzing primary human AML patient samples, the current study unexpectedly revealed that AML stem cells driven by MLL fusions almost exclusively resided in immunophenotypically mature CD34-/loCD38+ compartments, as demonstrated by in vitro long-term culture initiating cells and in vivo limiting dilution xeno-transplantation assays into immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Using highly purified populations of hematopoietic stem/progenitor cells from normal human umbilical cord blood (UCB) to reconstruct of the human disease, we found that only human HSCs and common myeloid progenitors (CMPs), but not developmentally branched LMPPs and GMPs, could be disease initiating cells, transformed by MLL-fusions and induced leukemia in vivo, suggesting a key difference between human and mouse leukemia. As revealed by RNA-sequencing, LMPPs and GMPs failed to activate stem cell transcriptional programs and genes essential for MLL-leukemia including MEIS1 and HMGA2 in the early phase of transformation. MLL-fusions transformed HSCs and CMPs were immunophenotypically indistinguishable to leukemic cells of human MLL-AML patients, and also had an enrichment of AML stem cells in CD34-/loCD38+ compartment. Using machine learning, a specific gene signature could stratify patients into HSCs- and CMPs-derived AML, in which HSCs-derived AML showed a significantly poorer prognosis. To further investigate the biology of HSCs/CMPs-derived MLL-AML related to treatment responses, we subjected HSC/CMP MLL-AML leukemia with chemotherapeutic drugs and inhibitor of Bromodomain and Extra-Terminal motif (BET) currently used in AML trial. Interestingly, CMP-derived MLL-AML was treatment sensitive and PDX models transplanted with CMP-like AML samples could be largely cured by chemotherapy or BET inhibitor targeted therapy. In contrast, human HSCs-derived AML was highly resistant to the treatments. Strikingly, shRNA-mediated knockdown or pharmacological inhibition by fidaxomicin targeting ATP-binding cassette (ABC) transporters, ABCC3 that is highly expressed in HSCs-derived MLL-AML could re-sensitize the cells to the current chemotherapy. Together, the current study not only for the first time functionally identifies the origin of human MLL-AML stem cells, but also provides a new actionable venue for overcoming stem cells-associated treatment resistance by repositioning an anti-diarrhea drug, fidaxomicin currently available in the clinics. Disclosures Mufti: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cellectis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Peripheral Blood Cell Sorting Strategies for Transcriptomic Analysis in Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic neoplasm characterized by sustained peripheral blood (PB) monocytosis and an inherent risk for leukemic transformation. Clonal origins of the disease can be detected in hematopoietic progenitor cells (CD34+/CD38-), while the complete spectrum of mutational evolution can be seen in circulating monocytes (CD14+). Cell sorting strategies have been employed to select cells in CMML, and while there are adequate monocyte numbers in the PB, there are very few circulating progenitor cells. In addition, attrition related to the selection process significantly depletes primary cells available for biological experiments and multiomics studies such as RNA-seq, ChIP-seq, ATAC-seq, and DIP-seq. While single-cell methods may be able to overcome this challenge, bulk sequencing methods remain a robust and cost-effective approach. We hypothesized that, secondary to the stem cell origin of this disease and significant myeloproliferation, PB mononuclear cells (MNC) would provide comparable results with regards to transcriptomic analysis, in comparison to cell selection procedures. Methods: Peripheral blood obtained from 15 molecularly annotated patients with WHO-defined CMML was ACK-lysed and subjected to a Ficoll procedure for collection of MNC. MNC were left unsorted (n=5) or further selected for CD34+/CD38- (n=5) and CD14+ (n=5) using a fully automated RoboSep-S (StemCell Technologies) protocol. All samples were then subjected to bulk whole transcriptome shotgun sequencing (using Illumina TruSeq and an Illumina HiSeq 4000). After data quality control, counts of detectable transcripts were log2-normalized and Pearson's product-moment correlation coefficients were calculated to evaluate the correlation between the two cell-sorting strategies and unsorted cells in terms of detectable transcripts. To visualize sample differences log2-normalized transcripts counts were centered and scaled per gene for a select number of genes relevant to myeloid biology as well as a number of housekeeping genes. Results: Fifteen patients with WHO-defined CMML, median age 69 years (55-73 years), 66% male, were included. Next generation sequencing for somatic mutations was performed on PB MNC obtained at CMML diagnosis (Figure 1, top heatmap). Considering the small sample size, mutations were evenly distributed among groups with the exception of ASXL1 (higher frequency in CD14+ and CD34+/CD38- cells), ZRSR2 (higher frequency in unsorted cells), and TET2 (lower frequency in CD14+ cells). The three groups were also well matched with regards to other CMML-related variables such as WHO and FAB morphological subtypes, cytogenetic abnormalities, and risk stratification by the Mayo Molecular Model. Transcriptomic analysis revealed a strong positive correlation between the median number of log2-normalized detectable transcripts in unsorted cells and CD34+/CD38- cells (ρ = 0.96, p < 0.001, top scatterplot). Likewise, there was a strong positive correlation between the median number of log2-normalized detectable transcripts in unsorted cells and CD14+ cells (ρ = 0.91, p < 0.001, bottom scatterplot). The latter correlation was marginally lower, which was explained by increased global gene expression in 3 of the 5 CD14+ samples (bottom heatmap). Increased gene expression in these 3 samples involved key myeloid genes and housekeeping genes known to have stable expression across human tissues alike. In comparison to PB MNC, both cell sorting strategies resulted in significant depletion of primary cells required for other experiments, and for procedures such as ChIP-seq, DIP-seq and ATAC-seq (CD34+/CD38- had greater depletion than CD14+). Additional experiments to assess this strategy for the above mentioned epigenetic studies are currently being planned. Conclusions: Accounting for sample differences, different cell sorting strategies (unsorted, CD34+/CD38- selection, and CD14+ selection) yielded similar results when performing bulk transcriptomic assessments on PB MNC from patients with CMML. For the purpose of gene expression profiling there was no clear advantage with CD34+/CD38- or CD14+ selection. These results support the use of unsorted cells for bulk transcriptomic analysis in CMML. Figure 1 Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Profile of Myeloproliferative Neoplasms in Kuwait: Population-Based Study

Introduction Myeloproliferative neoplasms (MPNs) are a group of hematopoietic disorders of stem-cell origin, characterized by mutations that disrupt hematopoietic signal-transduction pathways. The Middle East lacks an MPN registry representative of the disease in our area. Here we report on the epidemiology of these neoplasms in our area, including phenotype, clinical features and relevant outcomes. Methods This population-based study reports various demographic characteristics and clinical attributes of all suspected and confirmed MPN patients from all over Kuwait referred to the research hematology lab at Kuwait University & cytogenetic lab in Kuwait Cancer Control Centre (KCCC) during the period from 2007 to 2018. Molecular determination of the patients' driver mutation status currently relies on ARMS-PCR. Confirming a diagnosis follows the WHO criteria, and its refinements, for the diagnosis of MPNs. Data entry and analysis was performed using SPSS (v.22) software. Results Most patients are ≥ 40 years old (79.8%), with a median age of 55 years. Gender distribution is almost equal, with ethnic categorization as Kuwaiti and Non-Kuwaiti showing a similar pattern. ET is the most common diagnosis (40.1%), followed by PRV (32.3%). JAK2 V617F mutation is reported positive in 89.7% of cases, followed by CALR in 8.0% of MPNs. The incidence of MPNs ranged from 0.5 to 2.1 per 100,000 in 2007 through 2018. The lowest rate was recorded in 2007 (0.511) and the highest was observed in 2011 and 2016 (2.417 and 2.101, respectively). The increase in 2011 is likely due to the introduction of a more sensitive technique using ARMS-PCR for the diagnosis of MPNs, whereas the increase in 2017 may be explained by the publication of WHO 2016 modified criteria. Moreover, throughout the years, the distribution of MPNs in different age groups showed similar pattern, with the highest incidence in patients aged ≥ 60. Driver mutations can fit with a general increase in incidence from 2007-2017, which may be attributed to increased awareness among treating physicians asking suspected cases to screen for MPNs using molecular techniques.One hundred and twenty-four (18.5%) cases were documented to have a prior history of thrombosis, with roughly equal distribution between arterial and venous sites. A large proportion (89.5%) of the thrombotic events occurred in those who are ≥ 40 years old, with most events being associated with ET (34.7%) and PRV (33.1%). Almost one-third of cases of thrombosis were associated with undetermined MPN diagnosis. Participating patients were categorized as either low or high risk for thrombotic events, with the latter being defined as age ≥ 55 years and the presence of a previous thrombotic event. The results demonstrate that a total of 46 cases were defined as high risk, most of them being associated with ET (20 cases) and PRV (19 cases). A statistically significant association was reported between gender and site of occurrence of thrombotic events, with males having more arterial thromboses, and females were documented to have more venous thromboses. Conclusion JAK2 V617F driver mutation is the most common positive finding in the participating patients. Roughly one-fifth of the participants encountered thrombotic events, and the site of thrombosis is associated with gender, demonstrating statistical significance. These results should warrant a more thorough evaluation of MPNs in Kuwait to provide a better understanding of its epidemiology. This can be achieved through optimized documentation of patients' data, and testing for additional novel driver mutations and transformation; as well as encourage physicians in primary care centers to refer suspected cases for molecular diagnosis. Disclosures No relevant conflicts of interest to declare.

An exceptional response to immunotherapy doublet in combined hepatocellular carcinoma-cholangiocarcinoma

Abstract Background We present here a case of a 67-year-old male. He presented in May 2018 with severe abdominal pain and weight loss of 25%. Alpha-feto protein (AFP) and Carbohydrate antigen 19-9 (CA19-9) were within normal limits, and CA125 was 5 times upper normal limit (ULN). Computed tomography showed liver masses and enlarged retroperitoneal lymph nodes. Biopsy from a liver mass showed Combined Hepatocellular Carcinoma (HCC)-Cholangiocarcinoma (CC) (CHC). This a rare tumor, with an incidence of less than 10% of primary liver tumors. In whole exome analysis, mutations suggested the bipotent cell origin and stem cell origin. There are no guidelines or randomized trials regarding treatment. In an analysis of 36 patients who were treated with chemotherapy, the progression-free survival was 2.8 months, with an overall response rate of 5.6%. Targeted agents had minimal effect on survival. Methods In our case, extensive genomic, transcriptomic and proteomic testing was performed. No genomic alterations were identified, tumor mutational burden was low and microsatellite status was stable. 7 of 9 immune checkpoint genes were overexpressed. A variant in CDK12 was noted, which was shown to be associated with elevated neoantigen burden and may predict benefit from immune checkpoint therapy. Results The patient began immunotherapy with ipilimumab and nivolumab followed by nivolumab, which he is continuing. The only side effects were hypothyroidism and Addison’s disease which are being treated. His clinical response was dramatic, he regained all the lost weight, and discontinued high dose opiate treatment. ECOG performance status improved from 3 to 0. Repeated PET-CT showed near complete response, ca125 decreased by 90% and liver function tests normalized. Table: 49P . Blood tests of case Before immunotherapy After 11 months of immunotherapy Normal range Alk Phos 456 83 38-150 GGT 588 72 15-73 Ca125 210 21 2-35 Conclusions We present here an exceptional case of a rare tumor, where the patient had a clinical, laboratory and radiological response and a significant improvement in quality of life, suggesting that these tumors are sensitive to immunotherapy. No published cases of this tumor have yet been treated with immunotherapy doublet. Legal entity responsible for the study The author. Funding Has not received any funding. Disclosure The author has declared no conflicts of interest.

Niche origin of mesenchymal stem cells derived microvesicles determines opposing effects on NSCLC: Primary versus metastatic

Novel therapeutic approaches that address the malignant cells in their stroma microenvironment are urgently needed in lung cancer. The stroma resident mesenchymal stem cells (MSCs) interact with cancer cells in diverse ways including microvesicles (MVs) that transfer proteins and RNA species thereby modulating recipient cells' phenotype. Previously, we have demonstrated that MSCs' secretome from the primary non-small cell lung cancer (NSCLC) niche (lung) and metastatic niche (bone marrow (BM)) demonstrate opposite effects on NSCLC cells in a translation initiation (TI) dependent manner. Here, we examined the effect of MVs secreted from BM-MSCs' or lung-MSCs (healthy, NSCLC) to NSCLC phenotype. Briefly, NSCLC cell lines treated with Lung or BM-MSCs' MVs were assayed for viability (WST-1), cell count/death (trypan), migration (scratch), TI status and MAPKs activation (immunoblotting). Corresponding to previous published trends, Lung-MSCs' MVs promoted NSCLC cells' assayed traits whereas, BM-MSCs' MVs suppressed them. Activation of MAPKs and autophagy was registered in lung-MSCs MVs treated NSCLC cell lines only. Furthermore, lung-MSCs' MVs' treated NSCLC cells demonstrated an early (5min) activation of MAPKs and TI factors (peIF4E/peIF4GI) not evident in BM-MSCs MVs treated cells. These observations depict a role for MSCs'-MVs in NSCLC phenotype design and display distinct differences between the primary and metastatic niches that correspond to disease progression.In conclusion, the systemic nature of MVs marks them as attractive therapeutic markers/targets and we propose that identification of specific cargoes/signals that differentiate between MSCs MVs of primary and metastatic niches may introduce fresh therapeutic approaches.

Cardiac Protection after Systemic Transplant of Dystrophin Expressing Chimeric (DEC) Cells to the mdx Mouse Model of Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a progressive lethal disease caused by X-linked mutations of the dystrophin gene. Dystrophin deficiency clinically manifests as skeletal and cardiac muscle weakness, leading to muscle wasting and premature death due to cardiac and respiratory failure. Currently, no cure exists. Since heart disease is becoming a leading cause of death in DMD patients, there is an urgent need to develop new more effective therapeutic strategies for protection and improvement of cardiac function. We previously reported functional improvements correlating with dystrophin restoration following transplantation of Dystrophin Expressing Chimeric Cells (DEC) of myoblast origin in the mdx and mdx/scid mouse models. Here, we confirm positive effect of DEC of myoblast (MBwt/MBmdx) and mesenchymal stem cells (MBwt/MSCmdx) origin on protection of cardiac function after systemic DEC transplant. Therapeutic effect of DEC transplant (0.5 × 106) was assessed by echocardiography at 30 and 90 days after systemic-intraosseous injection to the mdx mice. At 90 days post-transplant, dystrophin expression in cardiac muscles of DEC injected mice significantly increased (15.73% ± 5.70 –MBwt/MBmdx and 5.22% ± 1.10 – MBwt/MSCmdx DEC) when compared to vehicle injected controls (2.01% ± 1.36) and, correlated with improved ejection fraction and fractional shortening on echocardiography. DEC lines of MB and MSC origin introduce a new promising approach based on the combined effects of normal myoblasts with dystrophin delivery capacities and MSC with immunomodulatory properties. Our study confirms feasibility and efficacy of DEC therapy on cardiac function and represents a novel therapeutic strategy for cardiac protection and muscle regeneration in DMD.

Stem Cell Therapies: A Way to Promising Cures.

Stem cells carry the remarkable ability to differentiate into different cell types while retaining the capability to self-replicate and maintain the characteristics of their parent cells, referred to as potency. Stem cells have been studied extensively to better understand human development and organogenesis. Because of advances in stem cell-based therapies, regenerative medicine has seen significant growth. Ophthalmic conditions, some of which are leading causes of blindness worldwide, are being treated with stem cell therapies. Great results have also been obtained in the treatment of oral and maxillofacial defects. Stem-cell-based therapies have great potential in the treatment of chronic medical conditions like diabetes and cardiomyopathy. The unique property of stem cells to migrate towards cancer cells makes them excellent vectors for the transportation of bioactive agents or for targeting cancer cells, both primary and metastatic.While these therapeutic strategies are extremely promising, they are not without limitations. Failure to completely eradicate the tumor and tumor relapse are some of those concerns. Stem cells share some characteristics with cancer stem cells, raising concerns for increasing the risk of cancer occurrence. Ethical concerns due to the fetal origin of stem cells and cost are other major obstacles in the large-scale implementation of such therapies.

PTTG1: a Unique Regulator of Stem/Cancer Stem Cells in the Ovary and Ovarian Cancer.

Origin of cancer stem cells (CSCs) and mechanisms by which oncogene PTTG1 contributes to tumor progression via CSCs is not known. Ovarian CSCs exhibit characteristics of self-renewal, tumor-initiation, growth, differentiation, drug resistance, and tumor relapse. A common location of putative origin, namely the ovarian surface epithelium, is shared between the normal stem and CSC compartments. Existence of ovarian stem cells and their co-expression with CSC signatures suggests a strong correlation between origin of epithelial cancer and CSCs. We hereby explored a putative oncogene PTTG1 (Securin), reported to be overexpressed in various tumors, including ovarian. We report a previously overlooked role of PTTG1 as a marker of CSCs thereby modulating CSC, germline, and stemness-related genes. We further characterized PTTG1's ability to regulate (cancer) stem cell-associated self-renewal and epithelial-mesenchymal transition pathways. Collectively, the data sheds light on a potential target expressed during ovarian tumorigenesis and metastatically disseminated ascites CSCs in the peritoneal cavity. Present study highlights this unconventional, under-explored role of PTTG1 in regulation of stem and CSC compartments in ovary, ovarian cancer and ascites and highlights it as a potential candidate for developing CSC specific targeted therapeutics.

Neural Stem Cells of the Subventricular Zone as the Origin of Human Glioblastoma Stem Cells. Therapeutic Implications.

Human glioblastoma is the most aggressive type of primary malignant brain tumors. Standard treatment includes surgical resection followed by radiation and chemotherapy but it only provides short-term benefits and the prognosis of these brain tumors is still very poor. Glioblastomas contain a population of glioma stem cells (GSCs), with self-renewal ability, which are partly responsible for the tumor resistance to therapy and for the tumor recurrence after treatment. The human adult subventricular zone contains astrocyte-like neural stem cells (NSCs) that are probably reminiscent of the radial glia present in embryonic brain development. There are numerous molecules involved in the biology of subventricular zone NSCs that are also instrumental in glioblastoma development. These include cytoskeletal proteins, telomerase, tumor suppressor proteins, transcription factors, and growth factors. Interestingly, genes encoding these molecules are frequently mutated in glioblastoma cells. Indeed, it has been recently shown that NSCs in the subventricular zone are a potential cell of origin that contains the driver mutations of human glioblastoma. In this review we will describe common features between GSCs and subventricular zone NSCs, and we will discuss the relevance of this important finding in terms of possible future therapeutic strategies.

Surgical Treatment of Periodontal Diseases—a Review of Current Clinical Research

This manuscript reviews the literature concerning the surgical treatment of periodontal diseases focusing on studies published within the last three years (2016–2019). A thorough review of four databases was completed including clinical human research studies and systematic reviews. Several recent studies have focused on evaluating the effects of combining alloplastic materials with autologous platelet-rich products and growth factors. The results of most of these studies are similar to those with traditionally used allografts. New membranes of human, animal, stem cell, or alloplastic origin, used for guided tissue regeneration procedures, perform similarly to the traditionally used resorbable collagen membranes. Addition of pharmacologic agents to regenerative procedures presents moderately favorable results. Tissue engineering and stem cell research are also expanding, although most of these studies are in vitro or in animal models. Newer minimally invasive surgical techniques and equipment that allow for better visibility and less invasive surgical approach have shown favorable results. Finally, systematic reviews conclude that there is lack of evidence to support LASERs as an alternative to more traditional surgical periodontal treatment. The focus of many recent human clinical studies has been to evaluate the combination of alloplastic and xenogeneic materials with autologous platelet-rich products, growth factors, and pharmacologic agents. The results of these combinations are similar to traditionally used allografts. The great variability between study design, materials, and techniques highlights the need for well-designed randomized controlled trials and longitudinal studies.

Breast Cancer Stem Cells and Sex Steroid Hormones.

Breast cancer stem cells (BCSCs) are a small population of tumor-initiating cells that express stem cell-associated markers. In recent years, their properties and mechanisms of regulation have become the focus of intense research due to their intrinsic resistance to conventional cancer therapies. This review describes breast cancer stem cell origin, signaling pathways involved in self-renewal, such as Wnt, Notch and Hedgehog, biomarkers linked to stemness, and the role of sex steroid hormones in BCSC regulation.

Abstract 1168: Characterization of tumor initiation cells in mammary tissues of C3-1-TAg transgenic mice

Abstract Animal models are powerful tools for the understanding of tumor development and cancer stem cell (CSC) origin. C3-1-TAg transgenic mice are a model for human triple negative breast cancer. Although general patterns of tumor development in this model have been well characterized, the tumor initiation cells of this model have not been defined. The objective of this study was to characterize the stemness and growth properties of mammary tumor cells in context with mammary epithelial cell (MEC) subpopulations of C3-1-TAg transgenic mice to identify the origin of tumor initiation cells. We first isolated MECs from mammary tissues of C3-1-TAg mice at various ages (8, 17, and 23 weeks old) and primary tumor cells from tumors of this model. Flow cytometry analysis with CD24 and CD49f as cell markers showed that cells from mammary tissues were separated into three subpopulations, including luminal, basal, and stromal populations. We found that the percentage of luminal subpopulation increased with age. Analysis of tumor derived cells using the same markers indicated that the majority of the cells in the plots overlapped with the position of luminal epithelial cells, suggesting their mutual connections. Next, we performed FACS sorting of mammary epithelial cells from the glands based on CD24 and CD49f and obtained four subpopulations of cells, including P1(stromal), P2(luminal), P3(CD24high/CD49fhigh, top of basal population on the plot), and P4(CD24mid/CD49fhigh, lower part of basal population), followed by the characterization of each subpopulations. Colony formation cell (CFC) assay and mammosphere assay were used for evaluation of luminal progenitor cells and self-renewal potential, respectively. Data from CFC assays showed that CFC colony numbers were the highest from the P2 population, followed by a few colonies from P3 population. Mammosphere assay showed that cells from P1 population formed typical mammospheres, while as cells from P2 and P3 formed cellular clusters different from typical mammospheres. FACS sorting with cells from C3-1-TAg mammary tumors obtained P2 and P3 subpopulations. Cells from P2, not P3, subpopulation were efficient in mammosphere formation. Because cells in P2 population are luminal mammary epithelial cells and are enriched with luminal progenitor cells, integration of data above suggest that C3-1-TAg mammary tumor cells are mainly derived from luminal progenitor cells. Using CD61, which is a luminal progenitor cell marker, we found that the percentage of luminal progenitor cells (CD61+/CD49fhigh) in the mammary glands was increased with age, and most tumor derived cells were CD61+/CD49fhigh. Taken together, our data demonstrate that the tumor initiation cells in the C3-1-TAg mammary tumors were derived from luminal progenitor cells. These findings lay a foundation for further characterization of cellular basis and molecular analysis of tumorigenesis in C3-1-TAg transgenic models. Citation Format: Qiong Cheng, Amanda B. Parris, Zhikun Ma, Yujie Shi, Lingfei Kong, Xiaohe Yang. Characterization of tumor initiation cells in mammary tissues of C3-1-TAg transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1168.

Abstract 5205: EWS-FLI1 activates HOXD13 expression through enhancer reprogramming

Abstract Ewing sarcomas (ES) are bone tumors of mesenchymal stem cell (MSC) origin. ES is defined by EWS-FLI1 fusions, which initiate tumorigenesis via enhancer reprogramming. Posterior homeobox D (HOXD) genes, in particular HOXD13, are over-expressed by ES, where they function as obligate, cooperative oncogenes. These genes play crucial roles in limb development and spatiotemporal control of HOXD13 expression depends on long-range enhancers that flank the HOXD locus. We hypothesize that HOXD13 is aberrantly activated in the ES cell of origin as a result of EWS-FLI1-dependent hijacking of developmental enhancers. We interrogated ChIP-seq and RNA-seq data to identify EWS-FLI1 binding sites and HOXD13 activation states in ES cells. These analyses revealed an EWS-FLI1 binding site within the developmentally conserved posterior HOXD enhancer region, which we will refer to as Desert Enhancer 268 (DE268). These sites were also marked with the active enhancer marks, H3K27Ac and H3K4me1. EWS-FLI1 knockdown was associated with loss of EWS-FLI1 binding, loss of H3K27Ac and H3K4me1, and loss of HOXD13 expression. Findings were independently validated by ChIP-qPCR and RT-qPCR. Through BruUV-seq we also identified that DE268 has eRNA expression that is lost upon EWS-FLI1 knockdown. Thus, HOXD13 expression in ES cells is maintained by EWS-FLI1-mediated enhancer activation. To address whether hijacking of HOXD13 enhancers is an initiating event in tumorigenesis, we evaluated enhancer profiles in MSCs ± EWS-FLI1. Whereas DE268 was not active in parent MSC, EWS-FLI1 binding and enhancer activation were evident in EWS-FLI1+ MSC. However, although EWS-FLI1 reproducibly binds and activates DE268, expression of HOXD13 is limited to discrete conditions. Together, these results suggest that EWS-FLI1 is necessary for enhancer activation, but that the fusion is alone is insufficient for induction of HOXD13 expression. Ongoing studies will utilize CRISPRi technologies to determine the contribution of DE268 on HOXD13 gene expression. Citation Format: April Ann Apfelbaum, Laurie Svoboda, Brian Magnuson, Mats Ljungman, Elizabeth Lawlor. EWS-FLI1 activates HOXD13 expression through enhancer reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5205.

Abstract 3682: Therapeutic targeting of stem cell self-renewal in childhood medulloblastoma: Strategies for blocking recurrence

Abstract Introduction: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Group 3 MB patients face the highest incidence of metastasis and poor overall patient survival. The early onset and highly aggressive nature of MB suggest a stem cell origin, where a highly self-renewing transformed cell of the postnatal cerebellum drives MB tumorigenesis. In this work, we explore how WNT signaling and other essential drivers of self-renewal, BMI1 and MSI1, promote MB progression. We subsequently generate new strategies to therapeutically target mechanisms of MB stem cell self-renewal that drive treatment resistance and relapse in Group 3 MB. Experimental procedures: We apply stem cell assays, patient-derived human-mouse xenograft (PDX) models, and genomic and bioinformatic profiling of recurrent patient-derived MB. Our established brain tumor initiating cell (BTIC) model provides an excellent tool for the examination of developmental pathways implicated in MB. New Unpublished Data: A small molecule Bmi1 inhibitor, PTC-028, induced a remarkable decrease in self-renewal as well as reduction of local and spinal metastatic disease in recurrent MB, which is striking as no prior drug has shown efficacy against recurrent Group 3 MB. Although mouse and human neural stem cells (NSCs) express Bmi1 and are mildly sensitive to Bmi1 inhibitors, no significant toxicity was observed in either mouse or human NSCs upon PTC-028 treatment, at doses that induced efficacious killing of MB cells. Another novel therapeutic paradigm includes activating Wnt signaling in otherwise non-Wnt MB, which abrogates self-renewal and tumorigenicity of these highly aggressive tumors. For safe and non-toxic activation of Wnt in preclinical models, we identified L807mts, a novel inhibitor that functions through a substrate-to-inhibitor conversion mechanism within the catalytic site of GSK. A final therapeutic strategy to target self-renewal lies in the discovery of the targetable MB-specific interactome of the RNA binding protein (RBP) Musashi1, another key regulator of stem cell self-renewal. Msi1 is overexpressed in Group 3 MB compared to normal cerebellum, and is associated with poor patient prognosis. shRNA knockdown of Msi1 decreased the self-renewal capacity of MB stem cells and significantly decreased tumor burden and increased survival in our PDX model. Finally, comparative eCLIP (enhanced cross-linking and immunoprecipitation) of MB stem cells and normal NSCs, combined with mass spectrometry and RNA-sequencing of shMsi1 MB cells, has elucidated novel therapeutic targets in the RBP interactome of Msi1 Conclusion: Characterization and therapeutic targeting of self-renewal mechanisms unique to MB BTICs may provide an opportunity to limit treatment-resistant stem cell populations from driving patient relapse in recurrent Group 3 MB, a disease currently lacking any targeted therapies. Note: This abstract was not presented at the meeting. Citation Format: David Bakhshinyan, Michelle Kameda-Smith, Branavan Manoranjan, Ashley Adile, Chitra Venugopal, Sheila Kumari Singh. Therapeutic targeting of stem cell self-renewal in childhood medulloblastoma: Strategies for blocking recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3682.

Маркеры стволовых клеток и их прогностические значения в уротелиальных карциномах мочевыделительной системы

Our aim was to carry out a comparative analysis of the expression of stem markers in the molecular subtypes of urothelial carcinomas, including ALDH1A1, CXCR4, CD24, CD82, CD105, CD133, NANOG, OCT4 and SOX-2. In addition, the relationship between the pattern of expression and the pathological features of the tumor was determined. Surgical specimens from 196 patients with a diagnosis of urothelial carcinoma of the renal pelvis and bladder were studied. Immunohistochemical study was performed on paraffin sections using the standard protocol. Antibodies against ALDH1A1, CD82, CD133, CXCR4, NANOG, OCT4, SOX2 ("Abcam"), CD24, CD105 ("Invitrogen"), CD31, CD34 ("Novocastra") were used. The stem cell markers used in the study were expressed in all molecular subtypes of urothelial carcinoma and there were no differences in frequency and intensity of expression between different phenotypes. However, the frequency and intensity of expression of the markers correlated with the tumor stage and the grade of cellular anaplasia. Our results confirm that cancer stem cells with basal phenotype are not an exclusive subpopulation in urothelial tumors. Other progenitor cells with the immunophenotype of intermediate and/or umbrella cells can serve as cancer stem cells. These features of the expression in cancer stem cell markers will allow to develop new approaches to the treatment of urothelial carcinomas.

Origins of intestinal stem cells - all in it together?

Origins of intestinal stem cells - all in it together?

PB1990 THE CHANGE OF BONE MARROW MICROENVIRONMENT OF SECONDARY POOR GRAFT FUNCTION AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION AND ITS EFFECT ON PROGNOSIS

Background:Secondary poor graft functionis a serious life‐threatening complication after allogeneic hematopoietic stem cell transplantation, which pathogenesis, evolution and prognosis are still unclear.Aims:To explore the role of adhesion related indicators in secondary poor graft functionafter allogeneic hematopoietic stem cell transplantationMethods:In this study, clinical data and post‐transplantation status of 106 patients with hematological tumour who were enrolled in our center and underwent allo‐HSCT from Jan. 1, 2016 to Jan. 31, 2017 was counted.The patients were divided into good graft function (GGF) group and secondary poor graft function (sPGF) group according to the implantation status after transplantation. The differences of age, primary disease, stem cell source, HLA matching, sex matching, ABO blood group matching, degree of bone marrow hyperplasia before transplantation, recurrence rate and mortality between the two groups were compared.29 patients with sPGF after transplantation were selected as the experimental group while 29 patients with GGF were selected as the control group.Results:The changes of adhesion related indicators in the two groups were detected.There were no significant differences in age, primary disease, stem cell origin, HLA matching, sex matching, ABO blood group matching and the degree of bone marrow proliferation before transplantation between the two groups (P > 0.05). The infection rate of CMV in sPGF group was significantly higher than that in GGF group, and the remission rate before transplantation in sPGF group was significantly lower than that in GGF group (P < 0.05).Cox regression analysis showed that the survival rate of patients with sPGF after allo‐HSCT was significantly lower than that of patients without sPGF (P < 0.05, HR = 3.598).The expression of CD44, CD29, ICAM‐1 and VCAM‐1 in sPGF group was not significantly different from that in control group (P > 0.05), but the expression of CD105 was higher than that in control group, and the expression of Cx43 was lower than that in control group (P < 0.05).Summary/Conclusion:We believe that the survival rate of patients with sPGF after allo‐HSCT will decrease significantly, and the occurrence of sPGF after transplantation may be related to the increase of CD105 expression and the decrease of Cx43 expression in bone marrow stromal cells before transplantation.

Individual brain organoids reproducibly form cell diversity of the human cerebral cortex.

Summary ParagraphExperimental models of the human brain are needed for basic understanding of its development and disease1. Human brain organoids hold unprecedented promise for this purpose; however, they are plagued by high organoid-to-organoid variability2,3. This has raised doubts as to whether developmental processes of the human brain can occur outside the context of embryogenesis with a degree of reproducibility comparable to the endogenous tissue. Here, we show that an organoid model of the dorsal forebrain can achieve reproducible generation of a rich diversity of cell types appropriate for the human cerebral cortex. Using single-cell RNA sequencing of 166,242 cells isolated from 21 individual organoids, we find that 95% of the organoids generate a virtually indistinguishable compendium of cell types, through the same developmental trajectories, and with organoid-to-organoid variability comparable to that of individual endogenous brains. Furthermore, organoids derived from different stem cell lines show consistent reproducibility in the cell types produced. The data demonstrate that reproducible development of complex central nervous system cellular diversity does not require the context of the embryo, and that establishment of terminal cell identity is a highly constrained process that can emerge from diverse stem cell origins and growth environments.

Stem Cells and Endometrial Regeneration: From Basic Research to Clinical Trial

Monthly changes in the endometrial cycle indicate the presence of endometrial stem cells. In recent years, various stem cells that exist in the endometrium have been identified and characterized. Additionally, many studies have shown that Bone Marrow Mesenchymal Stem Cells (BM-MSCs) provide an alternative source for regenerating the endometrium and repairing endometrial injury. This review discusses the origin of endometrial stem cells, the characteristics and main biomarkers among five types of putative endometrial stem cells, applications of endometrium-derived stem cells and menstrual blood-derived stem cells, the association between BM-MSCs and endometrial stem cells, and progress in repairing endometrial injury.