Impaired Differentiation Potential Research Articles

Nicotine-induced Genetic and Epigenetic Modifications in Primary Human Amniotic Fluid Stem Cells.

Smoking during pregnancy has been linked to adverse health outcomes in offspring, but the underlying mechanisms are not fully understood. To date, the effect of maternal smoking has been tested in primary tissues and animal models, but the scarcity of human tissues limits experimental studies. Evidence regarding smoking-related molecular alteration and gene expression profiles in stem cells is still lacking. We developed a cell culture model of human amniotic fluid stem cells (hAFSCs) of nicotine (NIC) exposure to examine the impact of maternal smoking on epigenetic alterations of the fetus. NIC 0.1 μM(equivalent to "light" smoking, i.e., 5 cigarettes/day) did not significantly affect cell viability; however, significant alterations in DNA methylation and N6-methyladenosine (m6A) RNA methylation in hAFSCs occurred. These epigenetic changes may influence the gene expression and function of hAFSCs. Furthermore, NIC exposure caused time-dependent alterations of the expression of pluripotency genes and cell surface markers, suggesting enhanced cell stemness and impaired differentiation potential. Furthermore, NICtreated cells showed reduced mRNA levels of key adipogenic markers and hypomethylation of the promoter region of the imprinted gene H19 during adipogenic differentiation, potentially suppressing adipo/lipogenesis. Differential expression of 16 miRNAs, with predicted target genes involved in various metabolic pathways and linked to pathological conditions, including cognitive delay and fetal growth retardation, has been detected. Our findings highlight multi-level effects of NIC on hAFSCs, including epigenetic modifications, altered gene expression, and impaired cellular differentiation, which may contribute to long-term consequences of smoking in pregnancy and its potential impact on offspring health and development.

Murine Myoblasts Exposed to SYUIQ-5 Acquire Senescence Phenotype and Differentiate into Sarcopenic-Like Myotubes, an In Vitro Study.

The musculoskeletal system is one of the most affected organs by aging that correlates well with an accumulation of senescent cells as for other multiple age-related pathologies. The molecular mechanisms underpinning muscle impairment because of senescent cells are still elusive. The availability of in vitro model of skeletal muscle senescence is limited and restricted to a small panel of phenotypic features of these senescent cells in vivo. Here, we developed a new in vitro model of senescent C2C12 mouse myoblasts that, when subjected to differentiation, the resulting myotubes showed sarcopenic features. To induce senescence, we used SYUIQ-5, a quindoline derivative molecule inhibitor of telomerase activity, leading to the expression of several senescent hallmarks in treated myoblasts. They had increased levels of p21 protein accordingly with the observed cell cycle arrest. Furthermore, they had enhanced SA-βgalactosidase enzyme activity and phosphorylation of p53 and histone H2AX. SYUIQ-5 senescent myoblasts had impaired differentiation potential and the resulting myotubes showed increased levels of ATROGIN-1 and MURF1, ubiquitin ligases components responsible for protein degradation, and decreased mitochondria content, typical features of sarcopenic muscles. Myotubes differentiated from senescent myoblasts cultures release increased levels of MYOSTATIN that could affect skeletal muscle cell growth. Overall, our data suggest that a greater burden of senescent muscle cells could contribute to sarcopenia. This study presents a well-defined in vitro model of muscle cell senescence useful for deeper investigation in the aging research field to discover new putative therapeutic targets and senescence biomarkers associated with the aged musculoskeletal system.

FAM122A Is Required for Mesendodermal and Cardiac Differentiation of Embryonic Stem Cells.

Mesendodermal specification and cardiac differentiation are key issues for developmental biology and heart regeneration medicine. Previously, we demonstrated that FAM122A, a highly conserved housekeeping gene, is an endogenous inhibitor of protein phosphatase 2A (PP2A) and participates in multifaceted physiological and pathological processes. However, the in vivo function of FAM122A is largely unknown. In this study, we observed that Fam122 deletion resulted in embryonic lethality with severe defects of cardiovascular developments and significantly attenuated cardiac functions in conditional cardiac-specific knockout mice. More importantly, Fam122a deficiency impaired mesendodermal specification and cardiac differentiation from mouse embryonic stem cells but showed no influence on pluripotent identity. Mechanical investigation revealed that the impaired differentiation potential was caused by the dysregulation of histone modification and Wnt and Hippo signaling pathways through modulation of PP2A activity. These findings suggest that FAM122A is a novel and critical regulator in mesendodermal specification and cardiac differentiation. This research not only significantly extends our understanding of the regulatory network of mesendodermal/cardiac differentiation but also proposes the potential significance of FAM122A in cardiac regeneration.

Bone marrow mesenchymal stromal cell-derived extracellular matrix displays altered glycosaminoglycan structure and impaired functionality in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of N-acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.

Impaired differentiation potential of CD34-positive cells derived from mouse hair follicles after long-term culture

Hair follicle epithelial stem cells (HFSCs), which exist in the bulge region, have important functions for homeostasis of skin as well as hair follicle morphogenesis. Although several methods for isolation of HFSCs using a variety of stem cell markers have been reported, few investigations regarding culture methods or techniques to yield long-term maintenance of HFSCs in vitro have been conducted. In the present study, we screened different types of commercially available culture medium for culturing HFSCs. Among those tested, one type was shown capable of supporting the expression of stem cell markers in cultured HFSCs. However, both the differentiation potential and in vivo hair follicle-inducing ability of HFSCs serially passaged using that optimal medium were found to be impaired, probably because of altered responsiveness to Wnt signaling. The changes noted in HFSCs subjected to a long-term culture suggested that the Wnt signaling-related environment must be finely controlled for maintenance of the cells.

CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

ABSTRACT It has recently been shown that CFIm25, a canonical mRNA 3’ processing factor, could play a variety of physiological roles through its molecular function in the regulation of mRNA alternative polyadenylation (APA). Here, we used CRISPR/Cas9-mediated gene editing approach in human embryonic stem cells (hESCs) for CFIm25, and obtained three gene knockdown/mutant cell lines. CFIm25 gene editing resulted in higher proliferation rate and impaired differentiation potential for hESCs, with these effects likely to be directly regulated by the target genes, including the pluripotency factor rex1. Mechanistically, we unexpected found that perturbation in CFIm25 gene expression did not significantly affect cellular mRNA 3’ processing efficiency and APA profile. Rather, we provided evidences that CFIm25 may impact RNA polymerase II (RNAPII) occupancy at the body of transcribed genes, and promote the expression level of a group of transcripts associated with cellular proliferation and/or differentiation. Taken together, these results reveal novel mechanisms underlying CFIm25ʹs modulation in determination of cell fate, and provide evidence that the process of mammalian gene transcription may be regulated by an mRNA 3’ processing factor.

ZFP451-mediated SUMOylation of SATB2 drives embryonic stem cell differentiation.

The establishment of cell fates involves alterations of transcription factor repertoires and repurposing of transcription factors by post-translational modifications. In embryonic stem cells (ESCs), the chromatin organizers SATB2 and SATB1 balance pluripotency and differentiation by activating and repressing pluripotency genes, respectively. Here, we show that conditional Satb2 gene inactivation weakens ESC pluripotency, and we identify SUMO2 modification of SATB2 by the E3 ligase ZFP451 as a potential driver of ESC differentiation. Mutations of two SUMO-acceptor lysines of Satb2 (Satb2K →R ) or knockout of Zfp451 impair the ability of ESCs to silence pluripotency genes and activate differentiation-associated genes in response to retinoic acid (RA) treatment. Notably, the forced expression of a SUMO2-SATB2 fusion protein in either Satb2K →R or Zfp451-/- ESCs rescues, in part, their impaired differentiation potential and enhances the down-regulation of Nanog The differentiation defect of Satb2K →R ESCs correlates with altered higher-order chromatin interactions relative to Satb2wt ESCs. Upon RA treatment of Satb2wt ESCs, SATB2 interacts with ZFP451 and the LSD1/CoREST complex and gains binding at differentiation genes, which is not observed in RA-treated Satb2K →R cells. Thus, SATB2 SUMOylation may contribute to the rewiring of transcriptional networks and the chromatin interactome of ESCs in the transition of pluripotency to differentiation.

TGFβ Family Signaling Pathways in Pluripotent and Teratocarcinoma Stem Cells’ Fate Decisions: Balancing Between Self-Renewal, Differentiation, and Cancer

The transforming growth factor-β (TGFβ) family factors induce pleiotropic effects and are involved in the regulation of most normal and pathological cellular processes. The activity of different branches of the TGFβ family signaling pathways and their interplay with other signaling pathways govern the fine regulation of the self-renewal, differentiation onset and specialization of pluripotent stem cells in various cell derivatives. TGFβ family signaling pathways play a pivotal role in balancing basic cellular processes in pluripotent stem cells and their derivatives, although disturbances in their genome integrity induce the rearrangements of signaling pathways and lead to functional impairments and malignant transformation into cancer stem cells. Therefore, the identification of critical nodes and targets in the regulatory cascades of TGFβ family factors and other signaling pathways, and analysis of the rearrangements of the signal regulatory network during stem cell state transitions and interconversions, are key issues for understanding the fundamental mechanisms of both stem cell biology and cancer initiation and progression, as well as for clinical applications. This review summarizes recent advances in our understanding of TGFβ family functions in naїve and primed pluripotent stem cells and discusses how these pathways are involved in perturbations in the signaling network of malignant teratocarcinoma stem cells with impaired differentiation potential.

MRNA Reprogramming of T8993G Leigh's Syndrome Fibroblast Cells to Create Induced Pluripotent Stem Cell Models for Mitochondrial Disorders.

Early molecular and developmental events impacting many incurable mitochondrial disorders are not fully understood and require generation of relevant patient- and disease-specific stem cell models. In this study, we focus on the ability of a nonviral and integration-free reprogramming method for deriving clinical-grade induced pluripotent stem cells (iPSCs) specific to Leigh's syndrome (LS), a fatal neurodegenerative mitochondrial disorder of infants. The cause of fatality could be due to the presence of high abundance of mutant mitochondrial DNA (mtDNA) or decline in respiration levels, thus affecting early molecular and developmental events in energy-intensive tissues. LS patient fibroblasts (designated LS1 in this study), carrying a high percentage of mutant T8993G mtDNA, were reprogrammed using a combined mRNA-miRNA nonviral approach to generate human iPSCs (hiPSCs). The LS1-hiPSCs were evaluated for their self-renewal, embryoid body (EB) formation, and differentiation potential, using immunocytochemistry and gene expression profiling methods. Sanger sequencing and next-generation sequencing approaches were used to detect the mutation and quantify the percentage of mutant mtDNA in the LS1-hiPSCs and differentiated derivatives. Reprogrammed LS-hiPSCs expressed pluripotent stem cell markers including transcription factors OCT4, NANOG, and SOX2 and cell surface markers SSEA4, TRA-1-60, and TRA-1-81 at the RNA and protein level. LS1-hiPSCs also demonstrated the capacity for self-renewal and multilineage differentiation into all three embryonic germ layers. EB analysis demonstrated impaired differentiation potential in cells carrying high percentage of mutant mtDNA. Next-generation sequencing analysis confirmed the presence of high abundance of T8993G mutant mtDNA in the patient fibroblasts and their reprogrammed and differentiated derivatives. These results represent for the first time the derivation and characterization of a stable nonviral hiPSC line reprogrammed from a LS patient fibroblast carrying a high abundance of mutant mtDNA. These outcomes are important steps toward understanding disease origins and developing personalized therapies for patients suffering from mitochondrial diseases.

Klotho Deficiency Accelerates Stem Cells Aging by Impairing Telomerase Activity.

Understanding the effect of molecular pathways involved in the age-dependent deterioration of stem cell function is critical for developing new therapies. The overexpression of Klotho (KL), an antiaging protein, causes treated animal models to enjoy extended life spans. Now, the question stands: Does KL deficiency accelerate stem cell aging and telomere shortening? If so, what are the specific mechanisms by which it does this, and is cycloastragenol (CAG) treatment enough to restore telomerase activity in aged stem cells? We found that KL deficiency diminished telomerase activity by altering the expression of TERF1 and TERT, causing impaired differentiation potential, pluripotency, cellular senescence, and apoptosis in stem cells. Telomerase activity decreased with KL-siRNA knockdown. This suggests that both KL and telomeres regulate the stem cell aging process through telomerase subunits TERF1, POT1, and TERT using the TGFβ, Insulin, and Wnt signaling. These pathways can rejuvenate stem cell populations in a CD90-dependent mechanism. Stem cell dysfunctions were largely provoked by KL deficiency and telomere shortening, owing to altered expression of TERF1, TGFβ1, CD90, POT1, TERT, and basic fibroblast growth factor (bFGF). The CAG treatment partially rescued telomerase deterioration, suggesting that KL plays a critical role in life-extension by regulating telomere length and telomerase activity.

Evaluation of Oxidative Stress and Mitophagy during Adipogenic Differentiation of Adipose-Derived Stem Cells Isolated from Equine Metabolic Syndrome (EMS) Horses.

Mesenchymal stem cells (MSCs) are frequently used in both human and veterinary medicine because their unique properties, such as modulating the immune response and differentiating into multiple lineages, make them a valuable tool in cell-based therapies. However, many studies have indicated the age-, lifestyle-, and disease-related deterioration of MSC regenerative characteristics. However, it still needs to be elucidated how the patient's health status affects the effectiveness of MSC differentiation. In the present study, we isolated mesenchymal stem cells from adipose tissue (adipose-derived mesenchymal stem cells (ASCs)) from horses diagnosed with equine metabolic syndrome (EMS), a common metabolic disorder characterized by pathological obesity and insulin resistance. We investigated the metabolic status of isolated cells during adipogenic differentiation using multiple research methods, such as flow cytometry, PCR, immunofluorescence, or transmission and confocal microscopy. The results indicated the impaired differentiation potential of ASCEMS. Excessive ROS accumulation and ER stress are most likely the major factors limiting the multipotency of these cells. However, we observed autophagic flux during differentiation as a protective mechanism that allows cells to maintain homeostasis and remove dysfunctional mitochondria.

NANOG restores the impaired myogenic differentiation potential of skeletal myoblasts after multiple population doublings

Adult skeletal muscle regeneration relies on the activity of satellite cells residing in the skeletal muscle niche. However, systemic and intrinsic factors decrease the myogenic differentiation potential of satellite cells thereby impairing muscle regeneration. Here we present data showing that late passage C2C12 myoblasts exhibited significantly impaired myogenic differentiation potential that was accompanied by impaired expression of myogenic regulatory factors (Myf5, MyoD, Myogenin, and MRF4) and members of myocyte enhancer factor 2 family. Notably, ectopic expression of NANOG preserved the morphology and restored the myogenic differentiation capacity of late passage myoblasts, possibly by restoring the expression level of these myogenic factors. Muscle regeneration was effective in 2D cultures and in 3D skeletal microtissues mimicking the skeletal muscle niche. The presence of NANOG was required for at least 15days to reverse the impaired differentiation potential of myoblasts. However, it was critical to remove NANOG during the process of maturation, as it inhibited myotube formation. Finally, myoblasts that were primed by NANOG maintained their differentiation capacity for 20days after NANOG withdrawal, suggesting potential epigenetic changes. In conclusion, these results shed light on the potential of NANOG to restore the myogenic differentiation potential of myoblasts, which is impaired after multiple rounds of cellular division, and to reverse the loss of muscle regeneration.

Dicer, a new regulator of pluripotency exit and LINE-1 elements in mouse embryonic stem cells.

A gene regulation network orchestrates processes ensuring the maintenance of cellular identity and genome integrity. Small RNAs generated by the RNAse III DICER have emerged as central players in this network. Moreover, deletion of Dicer in mice leads to early embryonic lethality. To better understand the underlying mechanisms leading to this phenotype, we generated Dicer‐deficient mouse embryonic stem cells (mESCs). Their detailed characterization revealed an impaired differentiation potential, and incapacity to exit from the pluripotency state. We also observed a strong accumulation of LINE‐1 (L1s) transcripts, which was translated at protein level and led to an increased L1s retrotransposition. Our findings reveal Dicer as a new essential player that sustains mESCs self‐renewal and genome integrity by controlling L1s regulation.

CBFβ-SMMHC Impairs Erythroid Differentiation and Induces Expansion of Aberrant Megakaryocytic/Erythroid Progenitors Capable of Leukemia Initiation

Approximately 12% of human acute myeloid leukemia (AMLs) harbor a recurrent chromosomal rearrangement inv(16)(p13q22). Inv(16) creates a fusion gene Cbfb-MYH11, encoding the fusion protein CBFß-SMMHC. Expressing CBFß-SMMHC in hematopoietic cells using a constitutive knock-in mouse model (Cbfb+/Cbfb-MYH11) or a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre; 129SvEv strain) causes defects in lymphoid and myeloid differentiation, and predisposes mice to AML. Previous studies with the constitutive knock-in mouse model showed impaired primitive erythropoiesis, however, Cbfb-MYH11 knocked-in cells were able to contribute to erythropoiesis in chimeric mice.To further delineate the effect of CBFß-SMMHC in adult erythropoiesis in the conditional knock-in mouse, we backcrossed Cbfb56M/+/Mx1-Cre into C57BL/6 and a Rosa26mT/mG Cre reporter strain. Induced expression of CBFß-SMMHC in adult mice leads to cell number dependent development of AML, consistent with previous studies in 129SvEv strain. Analysis of pre-leukemic bone marrow 2 weeks after induction revealed a 5.7-fold expansion of immunophenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E; Lin-cKit+Sca1-CD16-/loCD150+CD105-), and a 4.7 fold decrease of the erythroid progenitor (EP; Lin-cKit+Sca1-CD16-/loCD105hi) subset compared to similarly treated control mice. Both methylcellulose-based erythroid colony forming assay and in vitro erythroid differentiation culture showed that pre-leukemic Pre-Meg/Es expressing CBFß-SMMHC had an impaired differentiation potential for erythroid lineage. Using the Rosa26mT/mG Cre reporter allele, we tracked the proportions of CBFß-SMMHC- expressing cells (GFP+) in the Pre-Meg/E and EP subsets. We observed that the contribution of GFP+ cells sharply decreased in EPs but not in Pre-Meg/Es from primary pre-leukemic mice. Similar results were seen in transplant recipients engrafted with sorted GFP+ pre-leukemic Lin-cKit+Sca1+ cells. These results further confirmed that CBFß-SMMHC impairs cell-autonomous erythroid differentiation in vivo. Consistent with the impaired differentiation of Pre-Meg/Es, we observed altered expression pattern of erythroid regulatory genes, including Fog1, Gata2, and Gfi1b.The pre-leukemic Pre-Meg/Es exhibited increased colony forming and replating capacity in vitro and enhanced proliferation and survival in vivo. To determine whether these phenotypic Pre-Meg/E cells could be the cellular origin for leukemic transformation, we expressed a known cooperative onco-protein Mpl by retroviral transduction followed by transplantation. The majority of mice (83%) receiving 100,000 Pre-Meg/E cells developed leukemia with a medium onset of 92 days, suggesting that Pre-Meg/Es indeed are capable of leukemia initiation.In conclusion, the expression of CBFß-SMMHC impairs adult erythropoiesis at the transition of Pre-Meg/E to EPs, causing an expansion of Pre-Meg/E cells. These pre-leukemic Pre-Meg/Es could be the target cell of additional mutations contributing to leukemia transformation. DisclosuresNo relevant conflicts of interest to declare.

Abstract 177: Obesity promotes growth of ovarian cancer through adipose stem cells

Abstract Introduction: Intraperitoneal spread to visceral adipose is a frequent event and a major cause of morbidity and mortality in women with ovarian cancer. Excess visceral adiposity increases the risk of ovarian cancer. We hypothesized that obesity effects may be mediated by the adipose-derived stem cell population (ASC) to enhance the initiation and progression of ovarian cancer. Results: Intraperitoneally injected ID8 ovarian cancer cells grew significantly faster in mice with diet-induced obesity (p<0.05). To determine if ASC mediated this effect, ASC were then isolated from the subcutaneous (SC-ASC) and visceral adipose (V-ASC) of mice with diet-induced obesity and lean mice. ASC from all sources expressed same mesenchymal cell surface markers and similarly increased ID8 cell proliferation and survival following paclitaxel treatment. There was no significant difference in ID8 migration in response to ASC conditioned media from lean and obese mice. ASC from obese mice exhibited impaired differentiation into adipocytes and osteocytes in-vitro as compared to ASC from lean mice. 106 renilla luciferase expressing ID8 were co-injected intra-peritoneally with equal number of different ASC isolates into C57Bl6 mice. SC-ASC from obese mice promoted growth of intraperitoneal tumor and the formation of metastasis as compared to SC-ASC from lean mice (p<0.05). There was no significant difference in the effect of V-ASCs from obese and lean mice on ID8 cells. There was trend towards more rapid tumor growth in mice injected with V-ASC as compared to SC-ASC from lean mice. Ascites volume was highest in mice injected with SC-ASC derived from obese mice. SC-ASC from obese mice increased tumor vascularity while ASC from obese mice increased the formation of intra-tumoral perilipin expressing adipocytes. Conclusions: SC-ASC from obese mice promoted tumor growth more than ASC from lean mice suggesting that obesity alters the ASC population to enhance tumor-promoting effects. Obese derived ASC exhibit impaired differentiation potential, which may be linked to tumor promoting effects. Future studies will focus on understanding the mechanisms by which obesity alters the ASC population to impact tumor progression. Citation Format: Yan Zhang, Travis Solley, Karen Lu, Caimiao Wei, Alesandra Nowicka, Ann Klopp. Obesity promotes growth of ovarian cancer through adipose stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 177. doi:10.1158/1538-7445.AM2014-177

CEBPA Gene Different Mutants Play Distinct Impacts On Tumor Suppressor Function Of C/EBP-Alpha and Bone Marrow Stromal Cells

IntroductionCEBPA gene encodes CCAAT/enhancer-binding protein-alpha (C/EBPα), a crucial granulocytic differentiation factor and tumor suppressor in hematologic and non-hematologic malignancies. CEBPA gene is mutated in approximately 5–14% of acute myeloid leukemia (AML) patients and exhibited 3 different types of mutations, germ-line N-terminal mutation, N-terminal frameshift mutation and C-terminal mutation. Although murine models provide the functional consequences of CEBPA gene mutations in human hematopoiesis, the impacts of different mutations on abrogating C/EBPα functions and those of germ-line N-terminal mutations on bone marrow microenvironment have been received less attention. MethodsIn our previous research, we have provided the first report of multiple mutations of CEBPA contributing to the transformation of donor cells to the leukemic phenotype after allogeneic hematopoietic stem cell transplantation and identified 3 different CEBPA gene mutant forms which disrupted 3 major functional domains of C/EBPα respectively (H Xiao, Blood 2011; 117: 5257-5260). The patient and his donor-sister both harbored the N-terminal germ-line mutation (584_589dup disrupting the TAD2 domain of protein). Susceptible donor hematopoietic cells evolved to overt AML by developing two somatic CEBPA mutations, the N-terminal frameshift mutation (247dupC causing overproduction of truncated 30-KDa isoform (C/EBPα- p30) lacking the TAD1 domain) and the C-terminal mutation (914_916dup disrupting the bZIP domain), in the patient's microenvironment. We used these 3 mutant forms, as well as CEBPA gene wild type to subclone into pLenti6.3-MSC vector. Human leukemia cell lines (NB4, Kasumi-1, HL60, K562) and mouse myeloid progenitor cell line (32Dcl3) were transduced by different mutant forms to assess the impacts on blockage the major functions of C/EBPα including antileukemia effect and inducing granulocyte differentiation. To further discover previously unkown tumor suppressor genes dysregulated by C/EBPα mutant forms, the gene expression profiles of NB4 cells stably transfected with CEBPA gene different mutant forms were compared with that of CEBPA gene wild type vector by using Affymetrix PrimeView human gene expression microarray analysis. On the other hand, human normal bone marrow mesenchymal stromal cell ( MSC ) was transduced by N-terminal germ-line mutation to assess the impacts on the capacity of MSC to differentiate towards the osteogenic/adipogenic lineages, migrate and protect to leukemia cells. Results(1) The N-terminal germ-line mutation retains the functions of inducing apoptosis in leukemic cells and granulocyte differentiation of C/EBPα.The truncated C/EBPα-p30 protein mutant, produced from the N-terminal frameshift mutation, abrogates the effect of inducing apoptosis in leukemia cells. The C-terminal mutation (914_916 dup) abrogates both the effects of inducing apoptosis in leukemic cells and of promoting G-CSF-induced differentiation of 32Dcl3 cells into mature neutrophile granulocyte. (2) Gene expression microarray profiling analysis showed that compared with CEBPA gene wild type, leukemia-associated CEBPA somatic mutations, the N-terminal frameshift mutation and the C-terminal mutation, significantly inhibited the expression of ULBP2, an innate surface ligand of the natural killer (NK) cell receptor NKG2D, ultimately contributed to resistance to NK cell-mediated cytotoxicity. (3) MSCs harboring CEBPA N-terminal germ-line mutation showed impaired differentiation potential to osteogenic lineage by downregulation the expression of osteogenic genes (BSP1 and Runx2) , however similar in differentiation potential to adipogenic lineage, migration and the protection to leukemia cells, compared with MSCs with CEBPA wild type. ConclusionsOur data provide clues to support that the N-terminal frameshift mutation of CEBPA works as a class I mutation, while the C-terminal mutation works as both of class I and class II mutations in inducing leukemia. Furthermore, those leukemia-associated CEBPA somatic mutations abrogate the tumor suppressor function of C/EBPα by inhibiting NKG2D-mediated NK cell cytotoxicity. The N-terminal germ-line mutation retains the major functions of C/EBPα, however impaired the osteogenic differentiation potential of bone marrow stromal cells, which is important to support hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Aberrant Megakaryocytic/Erythroid Progenitors Contributes To Transformation Of Cbfb-SMMHC Induced Acute Myeloid Leukemia

Inv(16)(p13q22) is a recurrent chromosomal rearrangement found in approximately 12% of human acute myeloid leukemia (AML) cases and creates a fusion gene between CBFb and MYH11. The fusion gene encodes a fusion protein CBFß-SMMHC which causes defects in lymphoid and myeloid differentiation. Previous studies also showed that primitive erythropoiesis is impaired by CBFß-SMMHC, however, CBFß-SMMHC knocked-in cells was able to contribute to adult erythropoiesis in chimeric mice. Expressing CBFß-SMMHC in the hematopoietic cells using a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre; 129SvEv strain) recapitulates inv(16)-associated AML. Previous studies in this model showed that CBFß-SMMHC expression leads to pre-leukemic hematopoietic alterations, and together with additional cooperative mutations, result in spontaneous myeloid leukemia in mice with a 3-6 month latency. We hypothesized that an expanded cell population at the pre-leukemic stage could be the target of additional mutations, and hence the cell of origin of leukemia initiating cells.To further delineate the pre-leukemic progenitors and leukemia initiating cells, we backcrossed Cbfb56M/+/Mx1-Cre into C57BL/6 for more than 10 generations. Similar to previous studies in the129SvEv strain, expressing CBFß-SMMHC in adult C57BL/6 mice leads to cell number dependent development of AML. Analysis of pre-leukemic bone marrow as early as 2 weeks after induction revealed a 5.7-fold expansion of Pre-Meg/E cells (Pre-Megakaryocyte/Erythrocyte: Lin-cKit+Sca1-CD16-/loCD150+CD105-) compared to similarly treated control mice. While there was a significant increase in Pre-Meg/E population, we did not find significant increase in their proliferation but observed a 4.7 fold decrease of the erythroid progenitor (EP; Lin-cKit+Sca1-CD16-/loCD105hi) subset. Methylcellulose-based colony forming assay showed that pre-leukemic Pre-Meg/E had an impaired differentiation potential for erythroid lineage. In vitro erythroid differentiation assay also showed a partial block of differentiation from pre-leukemic Pre-Meg/E progenitors. These Pre-Meg/E like progenitors were able to induce leukemia in the presence of a known cooperative oncoprotein MPL when transplanted in lethally irradiated congenic recipient mice. In summary, our Results suggest that expression of CBFß-SMMHC impairs adult erythropoiesis at the transition of Pre-Meg/E to EPs, causing an expansion of Pre-Meg/E cells, which can be the target cell of additional mutations contributing to leukemia transformation. Disclosures:No relevant conflicts of interest to declare.

Amine‐surface‐modified superparamagnetic iron oxide nanoparticles interfere with differentiation of human mesenchymal stem cells

Superparamagnetic iron oxide (SPIO) nanoparticles have been widely used for stem cell labeling and tracking. Surface modification has been known to improve biocompatibility, biodistribution, and labeling efficiency of SPIO nanoparticles. However, the effects of amine (NH 3+)-surface-modified SPIO nanoparticles on proliferation and differentiation of human mesenchymal stem cells (hMSCs) remain unclear. The purpose of this study is to investigate how amine-surface-modified SPIO nanoparticles affected hMSCs. In this study, intracellular uptake and the contiguous presence of amine-surface-modified SPIO nanoparticles in hMSCs were demonstrated by Prussian blue staining, transmission electron microscopy and magnetic resonance imaging. Moreover, accelerated cell proliferation was found to be associated with cellular internalization of amine-surface-modified SPIO nanoparticles. The osteogenic and chondrogenic differentiation potentials of hMSCs were impaired after treating with SPIO, while adipogenic potential was relatively unaffected. Altered cytokine production profile in hMSCs caused by amine-surface-modified SPIO nanoparticles may account for the increased proliferation and impaired differentiation potentials; concentrations of the growth factors in the SPIO-labeled condition medium including amphiregulin, glial cell-derived neurotrophic factor, heparin-binding EGF-like growth factor and vascular endothelial growth factor, as well as soluble form of macrophage colony-stimulating factor receptor and SCF receptor, were higher than in the unlabeled-condition medium. In summary, although amine-surface-modified SPIO labeling is effective for cell tracking, properties of hMSCs may alter as a consequence and this needs to be taken into account when evaluating therapeutic efficacies of SPIO-labeled stem cells in vivo.

Satb1 and Satb2 regulate embryonic stem cell differentiation and Nanog expression

Satb1 and the closely related Satb2 proteins regulate gene expression and higher-order chromatin structure of multigene clusters in vivo. In examining the role of Satb proteins in murine embryonic stem (ES) cells, we find that Satb1(-/-) cells display an impaired differentiation potential and augmented expression of the pluripotency determinants Nanog, Klf4, and Tbx3. Metastable states of self-renewal and differentiation competence have been attributed to heterogeneity of ES cells in the expression of Nanog. Satb1(-/-) cultures have a higher proportion of Nanog(high) cells, and an increased potential to reprogram human B lymphocytes in cell fusion experiments. Moreover, Satb1-deficient ES cells show an increased expression of Satb2, and we find that forced Satb2 expression in wild-type ES cells antagonizes differentiation-associated silencing of Nanog and enhances the induction of NANOG in cell fusions with human B lymphocytes. An antagonistic function of Satb1 and Satb2 is also supported by the almost normal differentiation potential of Satb1(-/-)Satb2(-/-) ES cells. Taken together with the finding that both Satb1 and Satb2 bind the Nanog locus in vivo, our data suggest that the balance of Satb1 and Satb2 contributes to the plasticity of Nanog expression and ES cell pluripotency.

Lamin A/C and emerin are critical for skeletal muscle satellite cell differentiation

Mutations within LMNA, encoding A-type nuclear lamins, are associated with multiple tissue-specific diseases, including Emery-Dreifuss (EDMD2/3) and Limb-Girdle muscular dystrophy (LGMD1B). X-linked EDMD results from mutations in emerin, a lamin A-associated protein. The mechanisms through which these mutations cause muscular dystrophy are not understood. Here we show that most, but not all, cultured muscle cells from lamin A/C knockout mice exhibit impaired differentiation kinetics and reduced differentiation potential. Similarly, normal muscle cells that have been RNA interference (RNAi) down-regulated for either A-type lamins or emerin have impaired differentiation potentials. Replicative myoblasts lacking A-type lamins or emerin also have decreased levels of proteins important for muscle differentiation including pRB, MyoD, desmin, and M-cadherin; up-regulated Myf5; but no changes in Pax3, Pax7, MEF2C, MEF2D, c-met, and beta-catenin. To determine whether impaired myogenesis is linked to reduced MyoD or desmin levels, these proteins were individually expressed in Lmna(-/-) myoblasts that were then induced to undergo myogenesis. Expression of either MyoD or, more surprisingly, desmin in Lmna(-/-) myoblasts resulted in increased differentiation potential. These studies indicate roles for A-type lamins and emerin in myogenic differentiation and also suggest that these effects are at least in part due to decreased endogenous levels of other critical myoblast proteins. The delayed differentiation kinetics and decreased differentiation potential of lamin A/C-deficient and emerin-deficient myoblasts may in part underlie the dystrophic phenotypes observed in patients with EDMD.