Pluripotent Stem Cell Markers Research Articles

RNA-Sequencing Gene Expression Profiling of Orbital Adipose-Derived Stem Cell Population Implicate HOX Genes and WNT Signaling Dysregulation in the Pathogenesis of Thyroid-Associated Orbitopathy.

PurposeThe purpose of this study was to characterize the intrinsic cellular properties of orbital adipose-derived stem cells (OASC) from patients with thyroid-associated orbitopathy (TAO) and healthy controls.MethodsOrbital adipose tissue was collected from a total of nine patients: four controls and five patients with TAO. Isolated OASC were characterized with mesenchymal stem cell–specific markers. Orbital adipose-derived stem cells were differentiated into three lineages: chondrocytes, osteocytes, and adipocytes. Reverse transcription PCR of genes involved in the adipogenesis, chondrogenesis, and osteogenesis pathways were selected to assay the differentiation capacities. RNA sequencing analysis (RNA-seq) was performed and results were compared to assess for differences in gene expression between TAO and controls. Selected top-ranked results were confirmed by RT-PCR.ResultsOrbital adipose-derived stem cells isolated from orbital fat expressed high levels of mesenchymal stem cell markers, but low levels of the pluripotent stem cell markers. Orbital adipose-derived stem cells isolated from TAO patients exhibited an increase in adipogenesis, and a decrease in chondrogenesis and osteogenesis. RNA-seq disclosed 54 differentially expressed genes. In TAO OASC, expression of early neural crest progenitor marker (WNT signaling, ZIC genes and MSX2) was lost. Meanwhile, ectopic expression of HOXB2 and HOXB3 was found in the OASC from TAO.ConclusionOur results suggest that there are intrinsic genetic and cellular differences in the OASC populations derived from TAO patients. The upregulation in adipogenesis in OASC of TAO may be is consistent with the clinical phenotype. Downregulation of early neural crest markers and ectopic expression of HOXB2 and HOXB3 in TAO OASC demonstrate dysregulation of developmental and tissue patterning pathways.

192 Culture Conditions Supporting Long-Term Expansion of Bovine Spermatogonial Stem Cells Isolated from Adult and Immature Testes

Spermatogonial stem cells (SSC) self-renew and differentiate into spermatocytes to produce haploid sperm. Because SSC are a small population of adult stem cells in the testis, numerous studies have been reported to derive cell lines from cultured SSC. It has been reported that neonatal and adult mouse SSC can be cultured in vitro over the long term. Male germline stem (GS) cells, embryonic stem (ES)-like cells, and multipotent male germline stem (MGS) cells were derivated from mouse SSC. However, in domestic species including cattle, information about in vitro culture of SSC is mainly available in the neonatal and immature animal. To our knowledge, there are no reports about long-term culture of SSC isolated from adult bovine testis. In this report, we established culture conditions to maintain SSC isolated from adult and immature testes. The SSC were isolated by 3-step enzymatic digestion and enriched by Percoll gradient centrifugation. For adult testicular cell suspensions, SSC were further enriched by differential plating on precoated gelatin dish. After Percoll gradient centrifugation, we found differential expression of SSC markers (GFRα-1 and UCHL-1) in the isolated cells from immature and adult testis. The RT-PCR results also confirmed the expression of differentiated spermatogonia markers (SYCP3 and STRA-8) in adult testicular cell suspensions. It suggests that isolated testicular germ cell population from adult testis are more heterogeneous than those of immature testis. The SSC isolated from adult testes were cultured in low-serum media containing 6-bromoindirubin-3′-oxime (BIO), an inhibitor of glycogen synthase kinase-3α (GSK3), and subsequently the cultures were maintained in the medium containing glial cell line-derived neurotropic factor (GDNF). The cell lines have characteristics resembling mouse GS cell lines as confirmed by their grape-like shape morphology, the expression of SSC markers (UCHL-1, DBA, and GFRa-1), and pluripotent stem cell markers (POU5F1, SOX2, KLF4). The SSC from immature testes were proliferated for more than 3 months in serum-free culture conditions in the presence of GDNF and bovine leukemia inhibitory factor (LIF). The cell lines had ES-like cell morphology, expressed pluripotent stem cell markers and SSC-specific markers. They differentiated in vitro into 3 germ layers confirmed by the expression of ectoderm (NESTIN), mesoderm (BMP4), and endoderm (GATA-6) markers by RT-PCR and neuron like-cells confirmed by the expression of glial fibrillary acidic protein (GFAP) by immunofluorescence analysis. In conclusion, these findings indicate an efficient method to enrich SSC without cell sorting method and different long-term culture systems subsequently established to maintain SSC from adult and immature testes. Furthermore, our data would be useful for further studies that aim to preserve endangered species and improve livestock production through genome editing technology.

Single-cell mechanical phenotype is an intrinsic marker of reprogramming and differentiation along the mouse neural lineage.

Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells.

Mass spectrometry for identification of proteins that specifically bind to a distal enhancer of the Oct4 gene

Transcription factor Oct4 is a marker of pluripotent stem cells and has a significant role in their self-renewal. Oct4 gene is controlled by three cis-regulatory elements – proximal promoter, proximal enhancer and distal enhancer. All of these elements are targets for binding of regulatory proteins. Distal enhancer is in our research focus because of its activity in early stages of embryonic development. There are two main sequences called site 2A and site 2B that are presented in distal enhancer. For this moment proteins which bind to a site 2A (CCCCTCCCCCC) remain unknown. Using combination of in vitro method electrophoretic mobility shift assay (EMSA) and mass spectromery we identified several candidates that can regulate Oct4 gene expression through site 2A.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated β-galactosidase (SA-β-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal β-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry(IHC). The major limitation of the SA-β-Gal assay is the requirement of fresh or frozen samples. Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-β-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we useIHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Generation of an immortalized mesenchymal stem cell line producing a secreted biosensor protein for glucose monitoring.

Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations. Such devices use genetically modified cells expressing chimeric genes with glucose binding properties. MSCs are good candidates as carrier cells, as they can be genetically engineered and expanded into large numbers. They also possess immunomodulatory properties that, by reducing local inflammation, may assist long operation time.Here, we generated a novel immortalized human MSC line co-expressing hTERT and a secreted glucose biosensor transgene using the Sleeping Beauty transposon technology. Genetically modified hMSCs retained their mesenchymal characteristics. Stable transgene expression was validated biochemically. Increased activity of hTERT was accompanied by elevated and constant level of stem cell pluripotency markers and subsequently, by MSC immortalization. Furthermore, these cells efficiently suppressed PBMC proliferation in MLR transwell assays, indicating that they possess immunomodulatory properties. Finally, biosensor protein produced by MSCs was used to quantify glucose in cell-free assays. Our results indicate that our immortalized MSCs are suitable for measuring glucose concentrations in a physiological range. Thus, they are appropriate for incorporation into a cell-based, immune-privileged, glucose-monitoring medical device.

Embryo edit makes human 'knockout'.

CRISPR inactivation of gene allows developmental study.

Functional characterization of lysine-specific demethylase 2 (LSD2/KDM1B) in breast cancer progression.

Flavin-dependent histone demethylases govern histone H3K4 methylation and act as important chromatin modulators that are extensively involved in regulation of DNA replication, gene transcription, DNA repair, and heterochromatin gene silencing. While the activities of lysine-specific demethylase 1 (LSD1/KDM1A) in facilitating breast cancer progression have been well characterized, the roles of its homolog LSD2 (KDM1B) in breast oncogenesis are relatively less understood. In this study, we showed that LSD2 protein level was significantly elevated in malignant breast cell lines compared with normal breast epithelial cell line. TCGA- Oncomine database showed that LSD2 expression is significantly higher in basal-like breast tumors compared to other breast cancer subtypes or normal breast tissue. Overexpression of LSD2 in MDA-MB-231 cells significantly altered the expression of key important epigenetic modifiers such as LSD1, HDAC1/2, and DNMT3B; promoted cellular proliferation; and augmented colony formation in soft agar; while attenuating motility and invasion. Conversely, siRNA-mediated depletion of endogenous LSD2 hindered growth of multiple breast cancer cell lines while shRNA-mediated LSD2 depletion augmented motility and invasion. Moreover, LSD2 overexpression in MDA-MB-231 cells facilitated mammosphere formation, enriched the subpopulation of CD49f+/EpCAM- and ALDHhigh, and induced the expression of pluripotent stem cell markers, NANOG and SOX2. In xenograft studies using immune-compromised mice, LSD2-overexpressing MDA-MB-231 cells displayed accelerated tumor growth but significantly fewer lung metastases than controls. Taken together, our findings provide novel insights into the critical and multifaceted roles of LSD2 in the regulation of breast cancer progression and cancer stem cell enrichment.

Preliminary identification of endometrial cancer stem cells in vitro and in vivo

Stem cells play a critical role in endometrial cancer progression. However, the current methodologies used to isolate endometrial cancer stem cells (ECSCs) remain unsatisfactory. The ECSCs were isolated by serumfree suspension cultivation. The stem cells-related genes CD44, CD133, Oct4, Sox2 and Nanog were analyzed, and the biological behaviour of ECSCs was evaluated in vitro and vivo. The results suggest that (i) serumfree suspension cultivation is non-toxic and a convenient way for isolating the ECSCs, and is not limited to specific surface markers; (ii) Ishikawa cells can be used as an effective source of ECSCs, and the obtained ECSCs expressing the pluripotent stem cells markers CD44, CD133, Oct4, Sox2, and Nanog; (iii) ECSCs originated from Ishikawa cells showed an increased ability to invasion and metastasis in vitro, and exhibited a high proliferative capacity and pluripotency in vivo and vitro. These findings indicate that serumfree suspension cultivation is an effective method for isolating ECSCs from Ishikawa cells, and the obtained ECSCs are tumorigenic and display stem cell-like properties.

Murine melanoma cells incomplete reprogramming using non-viral vector.

The reprogramming of cancer cells into induced pluripotent stem cells or less aggressive cancer cells can provide a modern platform to study cancer-related genes and their interactions with cell environment before and after reprogramming. Herein, we aimed to investigate the reprogramming capacity of murine melanoma B16F10 cells. The B16F10 was transfected using non-viral circular DNA plasmid containing the genes Sox-2, Oct4, Nanog, Lin28 and green fluorescent protein (GFP). These cells were characterized by immunofluorescence, analysis RT-PCR and cell cycle. Our results demonstrated for the first time that reprogramming of B16F10 may be induced using non-viral minicircle DNA containing the four reprogramming factors Oct4, Sox2, Lin 28, Nanog (OSLN) and the GFP reporter gene. The resulting clones are composed by epithelioid cells. These cells display characteristics of cancer stem cells, thus expressing pluripotent stem cell markers and dividing asymmetrically and symmetrically. Reprogrammed B16F10 cells did not form teratomas; however, they showed the suppression of tumourigenic abilities characterized by a reduced tumour size, when compared with parental B16F10 cell line. In contrast to parental cell line that showed accumulation of the cells in S phase of cell cycle, the cells of reprogrammed clones are accumulated in G1 phase. Long-term cultivation of reprogrammed B16F10 cells induces regression of their reprogramming. Our data imply that in result of reprogramming of B16F10 cells less aggressive Murine Melanoma Reprogrammed Cancer Cells may be obtained. These cells represent an interesting model to study mechanism of cells malignancy as well as provide a novel tool for anti-cancer drugs screening.

Putative germline and pluripotent stem cells in adult mouse ovary and their in vitro differentiation potential into oocyte-like and somatic cells.

According to classical knowledge of reproductive biology, in the ovary of female mammals there is a limited number of oocytes and there is no possibility of renewal if the oocytes are lost due to disease or injury. However, in recent years, the results of some studies on renewal and formation of oocytes and follicles in the adult mammalian ovary have led to the questioning of this opinion. The aim of our study is to demonstrate the presence of putative germline and pluripotent stem cells in the adult mouse ovary and their differentiation potential into germ and somatic cells. In ovary tissues and cells harvested from pre-differentiation step, the expression of pluripotent and germline stem cell markers was analysed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and western blotting. Embryoid bodies that formed in this step were analysed using immunofluorescence staining and transmission electron microscopy. Ovarian stem cells were induced to differentiate into oocyte, osteoblast, chondrocyte and neural cells. Besides morphological observation, differentiated cells were analysed by RT-PCR, histochemical and immunofluorescence staining. Expression of germline and pluripotent stem cell markers both in mRNA and at the protein level were detected in the pre-differentiated cells and ovary tissues. As a result of the differentiation process, the formation of oocyte-like cells, osteoblasts, chondrocytes and neural cells was observed and characteristics of differentiated cells were confirmed using the methods mentioned above. Our study results revealed that the adult mouse ovary contains germline and pluripotent stem cells with the capacity to differentiate into oocyte-like cells, osteoblasts, chondrocytes and neural cells.

Generation of induced pluripotent stem cells from arrhythmogenic right ventricular disease patient

To establish the induced pluripotent stem cells (iPSCs) from patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). The fibroblasts were isolated from ARVC patient and DNA mutation sites were confirmed. We reprogrammed the patient fibroblasts to pluripotency by sendaiviral transduction with defined pluripotency factors. Then, we evaluated the pluripotency of ARVC-iPSCs by performing the immunofluorescence analysis, real-time PCR and 3 germ layer formation assay. We also induced the ARVC-iPSCs into cardiac specific differentiation by regulating Wnt signaling pathway. Apart from alkaline phosphatase positive staining, iPSCs derived from ARVC patients expressed pluripotent related genes and embryonic stem cell marker OCT4, SSEA4 and TRA-1-81. Embryonic body formation in vitro and teratoma test in vivo demonstrated that ARVC-iPSCs could differentiate into all three germ layers. After in vitro cardiac differentiation, ARVC-iPSC could be successfully induced to spontaneously beating mass with cTNT staining positive. Induced pluripotent stem cell was successfully established by integration free sendai virus from dermal fibroblast of patients with ARVC. It would provide the valuable experimental model to study the molecular mechanism of ARVC.

Development of a novel method for amniotic fluid stem cell storage

Background aimsCurrent procedures for collection of human amniotic fluid stem cells (hAFSCs) indicate that cells cultured in a flask for 2 weeks can then be used for research. However, hAFSCs can be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of this study was to determine whether direct freezing of amniotic fluid cells is able to maintain or improve the potential of a sub-population of stem cells. MethodsWe compared the potential of the hAFSCs regarding timing of freezing, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in a flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis and differentiation potential of C and D samples were compared. ResultshAFSCs isolated from D samples expressed mesenchymal stem cells markers until later passages, had a good proliferation rate and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, direct freezing induced a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. ConclusionsThis study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration, which affect stem cell properties. This technique might be a cost-effective and reasonable approach to the process of Good Manufacturing Process accreditation for stem-cell banks.

Stem‐Like Cells in Breastmilk for Hospitalized Preterm Infants

Breastmilk stem cells are a relatively new discovery in the scientific community. There are still many unknowns about their specific mechanisms of interaction; however, it has been hypothesized that their presence contributes to growth and development of the breastfed infant. Our research team is the first known team to enroll mothers of hospitalized preterm infants during the first few weeks of lactation and compare stem cell phenotypes and expression to mothers of healthy full‐term infants. The purpose of this study was to evaluate the feasibility of NICU breastmilk collection and the comparison of preterm and full‐term breastmilk cells. We enrolled mothers of preterm infants (n=7; range 26 4/7 – 34 5/7 weeks gestational age at birth) and mothers of healthy full‐term infants (n=4; range 39 5/7–40 5/7 weeks gestational age at birth). Flow cytometry and qRT‐PCR analysis were used to assess the prevalence of a variety of pluripotent and mesenchymal stem cell markers. Overall, cells with stem‐like characteristics were identified in both preterm and full‐term breastmilk samples. There was large variability among the amount of each stem cell phenotype present, as well as in the expression of specific markers. Preliminary results indicate that gene expression in the majority of the tested markers were significantly different between the preterm sample and the full‐term sample. In conclusion, there are individual differences in the cellular composition of breastmilk, and this variation results in differential expression of phenotypic markers and gene expression of breastmilk cells with multipotent and pluripotent characteristics. The science of breastmilk stem cells is new and there is future potential to use these easily accessible stem cells in regenerative medicine. Future studies will explore the relationship of breastmilk stem cell characteristics to neonatal health, most notably their regenerative and protective potential.Support or Funding InformationNational Association of Neonatal Nurses and Foundation for Neonatal Research and Education

Induced pluripotent stem cells derived from Bernard-Soulier Syndrome patient's peripheral blood cells with a p.Phe55Ser mutation in the GPIX gene

Bernard Soulier Syndrome (BSS) is a rare autosomal platelet disorder characterized by mutations in the von Willebrand factor platelet receptor complex GPIb-V-IX. In this work we have generated an induced pluripotent stem cell (BSS3-PBMC-iPS4F8) from peripheral blood mononuclear cells of a BSS patient with a p.Phe55Ser mutation in the GPIX gene. Characterization of BSS3-PBMC-iPS4F8 showed that these cells maintained the original mutation present in the BSS patient, expressed pluripotent stem cell markers and were able to differentiate into the three germline layers. This new iPSC line will contribute to better understand the biology of BSS disease.

The Pluripotent Stem-Cell Marker Alkaline Phosphatase is Highly Expressed in Refractory Glioblastoma with DNA Hypomethylation

Hypomethylation of genomic DNA induces stem-cell properties in cancer cells and contributes to the treatment resistance of various malignancies. To examine the correlation between the methylation status of stem-cell-related genes and the treatment outcomes in patients with glioblastoma (GBM). The genome-wide DNA methylation status was determined using HumanMethylation450 BeadChips, and the methylation status was compared between a group of patients with good prognosis (survival > 4 yr) and a group with poor prognosis (survival < 1 yr). Immunohistochemistry for proteins translated from hypomethylated genes, including alkaline phosphatase (ALPL), CD133, and CD44, was performed in 70 GBMs and 60 oligodendroglial tumors. The genomic DNA in refractory GBM was more hypomethylated than in GBM from patients with relatively long survival (P = .0111). Stem-cell-related genes including ALPL, CD133, and CD44 were also significantly hypomethylated. A validation study using immunohistochemistry showed that DNA hypomethylation was strongly correlated with high protein expression of ALPL, CD133, and CD44. GBM patients with short survival showed high expression of these stem-cell markers. Multivariate analysis confirmed that co-expression of ALPL + CD133 or ALPL + CD44 was a strong predictor of short survival. Anaplastic oligodendroglial tumors without isocitrate dehydrogenase 1 mutation were significantly correlated with high ALPL expression and poor survival. Accumulation of stem-cell properties due to aberrant DNA hypomethylation is associated with the refractory nature of GBM.

Ips Technology Revealed the Genetic and Functional Diversity Present in a Secondary AML Patient

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell diseases characterized by inefficient hematopoiesis and risk of progression to acute myeloid leukemia with poor prognosis. Although massive parallel sequencing studies have revealed a number of genomic alterations associated with myelodysplastic syndromes (MDS), the relationships between genetic architecture and clonal evolution in MDS remain poorly understood, mainly due to a difficulty in the ex vivo culture of primary MDS cells and a lack of good animal models. Induced pluripotent stem cells (iPSCs) from MDS patients are expected to provide a new platform for elucidation of the pathogenesis of MDS. We used reprogramming technology and transplantation into immunodeficient mice to functionally dissect the genetically defined subclones of secondary acute myeloid leukemia (AML) evolving from MDS (MDS/sAML).We attempted to generate iPSCs from bone marrow and peripheral blood mononuclear cells of a MDS/sAML patient with normal karyotype and multiple FLT3-internal tandem duplication (ITD) mutations, utilizing episomal methods. We successfully established more than 30 iPSC lines from sAML clones (sAML-iPSC) and 4 normal iPSC lines from normal clones at the same time. sAML-iPSC lines displayed characteristic morphology and expressed pluripotent stem cell markers at the levels comparable to those in isogenic normal iPSC lines and ES cell lines. Five randomly selected sAML-iPSC lines formed teratomas. Most of the sAML-iPSC lines retained normal karyotype even after 30 passages. About half of the sAML-iPSC lines harbored one out of four types of FLT3-ITDs identical to the primary sAML cells, reflecting the mosaicism of the FLT3-ITDs in the patient's sAML cells. Whole exome sequencing and SNP-CGH analysis revealed that all sAML-iPSC lines shared the chromosome 11p uniparental disomy (UPD) and 14 somatic single nucleotide variants (SNVs) identical to those of the primary sAML cells, indicating that these iPSC lines are derived from clonal sAML cells. Additionally, most of the sAML-iPSC lines had a frameshift mutation of CEBPA. Unexpectedly, 2 of the sAML-iPSC lines without any FLT3-ITD mutations had a FLT3-D835Y mutation, which was only detected by droplet digital PCR (ddPCR) at extremely low levels in the primary bone marrow sample.Hematopoietic differentiation efficiency of sAML-iPSC lines was comparable to that of isogenic normal iPSC lines. However, CD34+CD38-CD45+lineage- hematopoietic stem and progenitor cells (HSPCs) from sAML-iPSC lines displayed two to three times enhanced colony formation ability in myeloid-conditioning semifluid medium. Colonies formed by sAML-iPSC-derived HSPCs were predominantly composed of immature myeloblasts and could be replated for at least five rounds. Both FLT3-ITD- and FLT3-D835Y-positive sAML-iPSC-derived hematopoietic progenitor cells (HPCs) exhibited cytokine-independent growth but only FLT3-ITD-positive sAML cell lines retained in vitro sensitivity to AC220. To functionally evaluate the subclonal diversity in vivo, we performed xenotransplantation of HPCs from several sAML-iPSC lines into sublethally irradiated NSG mice. HPCs from all tested sAML-iPSC lines engrafted at 13 to 15 weeks after transplant. HPCs from FLT3-ITD-positive sAML-iPSC lines (n=5) displayed a higher ability to engraft and induced more severe sAML-like disease in recipient mice than those from FLT3-wild-type (wt) sAML-iPSC lines (n=4) (median spleen weight, 145.4 mg vs 55.7 mg; P < .05). Survival of mice injected with FLT3-ITD-positive sAML-iPSC-derived HPCs was shorter than those injected with FLT3-wt sAML-iPSC-derived HPCs (194 days vs 361 days; P< .001). To confirm the transformation properties of the FLT3-ITDs in this MDS/sAML patient, we transduced FLT3-wt sAML-iPSC-derived HPCs with retrovirus expressing a FLT3-ITD and transplanted the HPCs into recipient mice. FLT3-ITD-transduced recipients developed an aggressive sAML-like disease in 100% of recipient mice with shorter latency around 6 to 10 weeks, as evidenced by marked splenomegaly and massive expansion of immature myeloid cells in bone marrow and spleen.Our data suggest that this iPSC-based system could be useful for analysing clonal architecture and biological feature of subclones of MDS/sAML to identify candidate genes responsible for initiation and progression of MDS. DisclosuresTakaori-Kondo:Shionogi: Research Funding; Toyama Chemical: Research Funding; Mochida Pharmaceutical: Research Funding; Eisai: Research Funding; Cognano: Research Funding; Alexion Pharmaceuticals: Research Funding; Takeda Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau. Yamanaka:iPS Academia Japan: Consultancy.

Analysis of Leukemogenic Effects of RUNX1 and CSF3R Mutations Using Congenital Neutropenia (CN)/AML Patient-Derived Induced Pluripotent Stem Cells (iPSCs)

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome. Recently we reported a high frequency of cooperating RUNX1 and CSF3R mutations in CN patients that developed AML or MDS. Only a combination of these two mutations induced elevated proliferation and diminished myeloid differentiation of CD34+ cells in vitro.To confirm these clinical data in an in vitro model, we generated human induced pluripotent stem cells (hiPSCs) from PBMNCs of a CN patient harbouring p.C151Y mutation in ELANE after acquisition of AML. During GCSF treatment, this patient acquired G-CSFRmutation p.Q741*, which leads to a truncated G-CSF receptor and was detected six years prior to overt AML. Three years later, he acquired an additional RUNX1 (p.R139G) mutation, which is located in the RUNT-homology domain (RHD). Subsequently, he developed AML (FAB M1) with trisomy 21. Reprogramming of PBMNCs isolated from the time-point of AML (ca. 80 % of AML blasts) resulted in the generation of hiPSCs clones harbouring either only ELANE p.C151Y mutation (CN-iPSC clone, derived from non-leukemia PBMNCs) or additional CSF3R and RUNX1 mutations and trisomy 21 (CN/AML-iPSC clone, derived from AML blasts), which was subsequently validated by Sanger sequencing and by digital PCR.These iPSCs clones have been tested for their pluripotency and self-renewal capacity. Both iPSC clones expressed the pluripotent stem cell surface markers SSEA-4 and TRA-1-60 and displayed alkaline phosphatase activity. Further they highly expressed mRNA of the pluripotent stem cell markers SOX2, ABCG2, DNMT and NANOG and were able to differentiate into all three germ layers (meso-, endo- and ectoderm).Embryoid body (EB)-based hematopoietic / neutrophilic differentiation of CN-iPS clones using serum-free APEL stem cell differentiation medium showed comparable amounts of CD34+ and CD33+ cells, but ~ 2-fold reduction of CD16+ cells, compared to healthy donor (HD) iPSCs. CN/AML-iPSCs were not able to differentiate into mature granulocytes at all and revealed 10-fold reduced counts of CD34+ and CD33+hematopoietic cells. Morphological examinations of Giemsa-stained cytospin slides confirmed these results. Additionally, CN/AML-iPSCs showed a highly reduced number of CFU-G and CFU-GM colonies in CFU-Assay.To investigate the intracellular mechanisms of leukemogenic transformation in CN, we analyzed gene expression profiles of hematopoietic cells generated from CN-iPSCs vs CN/AML-iPSCs and HD-iPSCs for various time points of differentiation in our EB based-system. Our previous microarray-based analysis of bone marrow CD33+ cells of this CN/AML patient revealed that genes overexpressed in early hematopoietic stem/progenitor cells (HSPCs) as compared to more mature progenitors, such as DNTT, BAALC, CD34, HPGDS, NPR3 and PROM1 were strongly upregulated in CN/AML blasts harbouring both RUNX1 and CSF3R mutations, as compared to the cells prior to leukemia development. Intriguingly, elevated expression of these genes was described previously in RUNX1-mutated de novo AML blasts (Mendler et al., JCO 2012). This genetic signature suggests transformation of hematopoietic progenitors carrying mutated CSF3R into more primitive hematopoietic progenitors after aquisition of RUNX1mutation. We were able to confirm markedly increase of mRNA levels of these genes in hematopoietic cells derived from CN/AML-iPSCs, as compared to CN-iPSCs.In addition, we found that hematopoietic cells of both CN-iPSCs and CN/AML-iPSCs revealed increased expression of unfolded-protein response (UPR) genes DDIT3 (CHOP), ATF4 and ATF6, as compared to HD-iPSCs. Activation of UPR in hematopoietic cells of CN-ELANEpatients has been previously described by our and other groups. CN/AML-iPSC-derived hematopoietic progenitor cells expressed RUNX1 mRNA at least two-fold higher, as compared to HD- or CN-iPSC-derived cells.In summary, we established an in vitro cellular model of leukemogenic transformation in CN patients using CN/AML-patient derived hiPSCs that confirmed clinical data of Skokowa et al. (Blood 123:2550, 2014) on a cooperative leukemogenic effect of CSF3R and RUNX1 mutations. Comprehensive analysis of hematopoiesis using this iPSCs model will give us a deeper view into this highly complex signaling network operating during leukemogenic transformation of HSCs in pre-leukemic bone marrow failure syndromes. DisclosuresNo relevant conflicts of interest to declare.

Reprogramming of retinoblastoma cancer cells into cancer stem cells

Retinoblastoma is the most common intraocular malignancy in pediatric patients. It develops rapidly in the retina and can be fatal if not treated promptly. It has been proposed that a small population of cancer cells, termed cancer stem cells (CSCs), initiate tumorigenesis from immature tissue stem cells or progenitor cells. Reprogramming technology, which can convert mature cells into pluripotent stem cells (iPS), provides the possibility of transducing malignant cancer cells back to CSCs, a type of early stage of cancer. We herein took advantage of reprogramming technology to induce CSCs from retinoblastoma cancer cells. In the present study, the 4 Yamanaka transcription factors, Oct4, Sox2, Klf4 and c-myc, were transduced into retinoblastoma cells (Rbc51). iPS-like colonies were observed 15 days after transduction and showed significantly enhanced CSC properties. The gene and protein expression levels of pluripotent stem cell markers (Tra-1-60, Oct4, Nanog) and cancer stem cell markers (CD133, CD44) were up-regulated in transduced Rbc51 cells compared to control cells. Moreover, iPS-like CSCs could be sorted using the Magnetic-activated cell sorting (MACS) method. A sphere formation assay demonstrated spheroid formation in transduced Rbc51 cells cultured in serum free media, and these spheroids could be differentiated into Pax6-, Nestin-positive neural progenitors and rhodopsin- and recoverin-positive mature retinal cells. The cell viability after 5-Fu exposure was higher in transduced Rbc51 cells. In conclusion, CSCs were generated from retinoblastoma cancer cells using reprogramming technology. Our novel method can generate CSCs, the study of which can lead to better understanding of cancer-specific initiation, cancer epigenetics, and the overlapping mechanisms of cancer development and pluripotent stem cell behavior.

Expression of early transcription factors by mesenchymal stem cells derived from ovine umbilical cord Wharton’s jelly

Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into mesodermal lineages. Despite major progress in our general knowledge related to the application of adult stem cells, finding alternative sources for bone marrow MSCs has remained to be challenged. In this study, we utilized ovine umbilical cord Wharton’s jelly as a primary source for isolation of MSCs since it is a rich source of MSCs and no ethical issues were involved. Ovine umbilical cord Wharton’s jelly segments were digested enzymatically and cultured in vitro in culture medium. In addition to the study of their morphology and colony forming units, the expression of pluripotent stem cell markers by the isolated MSCs were also studied. The MSCs were plastic adherent, clonogenic and their morphology were polygonal, star shaped and fibroblast like. They revealed a strong expression of pluripotent stemness markers Oct4, Sox2, Nanog and Alkaline phosphatase. These cells confirmed their ability of self-renewal by expressing Sox2 gene and their properties of pluripotency and plasticity by expressing Oct4, Nanog and Alkaline phosphatase. The study revealed that Wharton’s jelly is a rich source of stem cells with stemness properties and mesenchymal like morphology and could be used as an alternate for the bone marrow derived MSCs for cell based regenerative therapies.