Progenitor Phenotype Research Articles

A KLF4-DYRK2–mediated pathway regulating self-renewal in CML stem cells

AbstractLeukemia stem cells are a rare population with a primitive progenitor phenotype that can initiate, sustain, and recapitulate leukemia through a poorly understood mechanism of self-renewal. Here, we report that Krüppel-like factor 4 (KLF4) promotes disease progression in a murine model of chronic myeloid leukemia (CML)-like myeloproliferative neoplasia by repressing an inhibitory mechanism of preservation in leukemia stem/progenitor cells with leukemia-initiating capacity. Deletion of the Klf4 gene severely abrogated the maintenance of BCR-ABL1(p210)–induced CML by impairing survival and self-renewal in BCR-ABL1+ CD150+ lineage-negative Sca-1+ c-Kit+ leukemic cells. Mechanistically, KLF4 repressed the Dyrk2 gene in leukemic stem/progenitor cells; thus, loss of KLF4 resulted in elevated levels of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2), which were associated with inhibition of survival and self-renewal via depletion of c-Myc protein and p53 activation. In addition to transcriptional regulation, stabilization of DYRK2 protein by inhibiting ubiquitin E3 ligase SIAH2 with vitamin K3 promoted apoptosis and abrogated self-renewal in murine and human CML stem/progenitor cells. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling p53- and c-Myc–mediated regulation of survival and self-renewal in CML cells with leukemic-initiating capacity that can be targeted with small molecules.

P4488Organotypic culture of the epicardium: a new tool for cardiovascular research

Abstract Background The epicardium, the most external layer of the heart, is composed of a layer of epithelial cells and underlying connective tissue. Following myocardial infarction, epicardial cells are activated and provide a source of paracrine factors and progenitor cells. In the border zone of the ischaemic tissue, the activated epicardial cells support cardiac and vascular regeneration by releasing pro-angiogenic and pro-survival factors, and by differentiating towards multiple cell lineages. During this process, activated epicardial cells migrate to the site of injury where they contribute to both post-ischemic remodelling and fibrosis. There is limited knowledge of the cellular and molecular regulation of these processes in large animals and humans, in part due to the lack of robust and representative models. Purpose In this project, we developed an ex vivo 3D organotypic model derived from porcine hearts, amenable to culture, which enables structural, molecular and cellular studies of the epicardium. Methods Thin epicardial/cardiac tissue slices (EpCardio-TS) were obtained by using a vibratome to cut the first layer of tissue from the epicardial side of porcine heart cubes. Slices were cultured for up to 72h in a bioreactor that uses a 3D printed chamber connected to a control system that allows maintenance and adjustment of culture conditions, and ensures continuous media flow. Local intracellular delivery of fluorescent quantum-dots (Qdots) was performed using nanoneedle chips to track epicardial cells, whilst cell fate is visualised in 3D by performing immunofluorescence on decolourised slices. Results Intact EpCardio-TS obtained from porcine heart included a viable epicardium, expressing typical epicardial markers (wt-1, mesothelin, uroplakin), and an electrically active myocardium. Live/dead staining showed epicardial (67.8±16.2%, N=5) and myocardial (40.8±28.6%, N=3) viability, and TUNEL assay confirmed low levels of apoptosis (6.3±5.1% of wt-1+ epicardial cells N=1). Moreover, the presence of proliferating epicardial cells (PCNA+), the increase in wt-1+ cells, and the increase in epicardial gene expression (Tbx18 and TCF21) suggested that cells maintain their progenitor phenotype and undergo activation in culture. Nanoinjection of fluorescent Qdots to EpCardio-TS localized them to the wt-1+ cells on the slice surface, presenting a strategy to mark the epicardial layer. This, combined with the successful decolourisation of the slices, provides an in vitro platform to track the role of epicardial cells in cardiac remodelling and fibrosis. Conclusions EpCardio-TS represents a robust ex vivo model merging the complexity of a 3D organotypic culture with the simplicity of the in vitro culture. EpCardio-TS are amenable to culture and cell tracking, and can therefore find application in toxicology and gene therapy screening for the modulation of epicardial interactions with myocardial and non-myocardial cells of the heart.

Noninvasive prediction of HCC with progenitor phenotype based on gadoxetic acid-enhanced MRI.

To explore the noninvasive prediction of hepatocellular carcinoma (HCC) with progenitor phenotype based on gadoxetic acid-enhanced magnetic resonance imaging (MRI). This retrospective study included 115 surgery-proven HCCs with preoperative gadoxetic acid-enhanced MRI from August 2015 to September 2018. Image features were reviewed. Quantitative image analysis was performed using histogram analysis. HCC with progenitor phenotype was defined as positive for either cytokeratin 19 (CK19) or epithelial cell adhesion molecule (EpCAM) expression. Statistically significant variables for identifying HCCs with progenitor phenotype were determined at multivariate analyses. ROC analyses were used to determined cutoff values and the diagnostic performance of significant variables and combinations. Prediction nomogram was constructed based on multivariate analysis. At multivariate regression analyses, AFP ≥ 155.25ng/mL (p < 0.001), skewness on T2WI ≤ 1.10 (p = 0.024), uniformity on pre-T1WI ≤ 0.91 (p = 0.024), irregular tumor margin (p = 0.006), targetoid appearance (p = 0.001), and the absence of mosaic architecture (p = 0.014) were significant predictors of HCCs expressing progenitor cell markers. Combing any three of those significant variables, it provides a diagnostic accuracy of 0.86 (95% CI 0.78-0.92) with sensitivity of 0.97 (95% CI 0.86-1.00), and specificity of 0.74 (95% CI 0.63-0.83). The C-index of the regression coefficient-based nomogram was 0.94 (95% CI 0.91-0.98). Noninvasive prediction of HCCs with progenitor phenotype can be achieved with high accuracy by integrated interpretation of biochemical and radiological information, representing a handy tool for precise patient management and the prediction of prognosis. • Qualitative image features of irregular tumor margin, targetoid appearance, and the absence of mosaic architecture are significant predictors of hepatocellular carcinoma with progenitor phenotype. • Quantitative analyses using whole-lesion histogram analysis provides additional information for the prediction of hepatocellular carcinoma with progenitor phenotype. • Noninvasive prediction of hepatocellular carcinoma with progenitor phenotype can be achieved with high accuracy by integrated interpretation of clinical information and qualitative and quantitative imaging analyses.

Discovery of retinoic acid receptor agonists as proliferators of cardiac progenitor cells through a phenotypic screening approach.

Identification of small molecules with the potential to selectively proliferate cardiac progenitor cells (CPCs) will aid our understanding of the signaling pathways and mechanisms involved and could ultimately provide tools for regenerative therapies for the treatment of post‐MI cardiac dysfunction. We have used an in vitro human induced pluripotent stem cell‐derived CPC model to screen a 10,000‐compound library containing molecules representing different target classes and compounds reported to modulate the phenotype of stem or primary cells. The primary readout of this phenotypic screen was proliferation as measured by nuclear count. We identified retinoic acid receptor (RAR) agonists as potent proliferators of CPCs. The CPCs retained their progenitor phenotype following proliferation and the identified RAR agonists did not proliferate human cardiac fibroblasts, the major cell type in the heart. In addition, the RAR agonists were able to proliferate an independent source of CPCs, HuES6. The RAR agonists had a time‐of‐differentiation‐dependent effect on the HuES6‐derived CPCs. At 4 days of differentiation, treatment with retinoic acid induced differentiation of the CPCs to atrial cells. However, after 5 days of differentiation treatment with RAR agonists led to an inhibition of terminal differentiation to cardiomyocytes and enhanced the proliferation of the cells. RAR agonists, at least transiently, enhance the proliferation of human CPCs, at the expense of terminal cardiac differentiation. How this mechanism translates in vivo to activate endogenous CPCs and whether enhancing proliferation of these rare progenitor cells is sufficient to enhance cardiac repair remains to be investigated.

Single cell analysis of human foetal liver captures the transcriptional profile of hepatobiliary hybrid progenitors

The liver parenchyma is composed of hepatocytes and bile duct epithelial cells (BECs). Controversy exists regarding the cellular origin of human liver parenchymal tissue generation during embryonic development, homeostasis or repair. Here we report the existence of a hepatobiliary hybrid progenitor (HHyP) population in human foetal liver using single-cell RNA sequencing. HHyPs are anatomically restricted to the ductal plate of foetal liver and maintain a transcriptional profile distinct from foetal hepatocytes, mature hepatocytes and mature BECs. In addition, molecular heterogeneity within the EpCAM+ population of freshly isolated foetal and adult human liver identifies diverse gene expression signatures of hepatic and biliary lineage potential. Finally, we FACS isolate foetal HHyPs and confirm their hybrid progenitor phenotype in vivo. Our study suggests that hepatobiliary progenitor cells previously identified in mice also exist in humans, and can be distinguished from other parenchymal populations, including mature BECs, by distinct gene expression profiles.

Abstract 859: Basal Notch4 activation is a druggable marker of aggressiveness in a panel of human cancer cell lines

Abstract Background: Notch pathway has been involved in cell differentiation, proliferation, apoptosis, angiogenesis and drug resistance, as well as in epithelial-to-mesenchymal transition. Notch pathway activation leads to a proteolytic cleavage, releasing the Notch intracellular domain (NICD), which translocates to the nucleus and activates target genes, such as HES1 (interacting with Notch for maintenance of stem cells and progenitor phenotype). Our team has demonstrated the involvement of PlGF/VEGFR1/Notch4 axis in the angiogenesis of hepatocellular carcinoma, but what about the role of Notch in other cancer types? The aim of this work is to characterize the basal activation and the role of Notch4 in a panel of human cancer cell lines. Materials and Methods: We characterized a panel of 8 pancreatic (PDAC), 8 head and neck (H&amp;N), 5 colorectal (CRC), 5 cholangiocarcinoma (CK), and 10 hepatocellular (HCC) human carcinoma cell lines for intracellular domain of the notch protein 4 (NICD4), HES1, E-cadherin, and Vimentin expression by Western Blot. In each tumor type, cell lines with high and low Notch4 activation were selected to assess basal cell proliferation and migration, using MTT and wound-healing assay, respectively. In high versus low Notch4 cell lines, we assessed NUMB expression (involved in NICD proteasomal degradation) and the effect of a Notch inhibitor, PF- 03084014. Results: Notch4 activation assessed by NICD4 expression was observed in 6 out of 8 (6/8) PDAC, 7/8 H&amp;N, 2/5 CRC, 4/5 CK, and 5/10 HCC cell lines. In PDAC cells, Notch4 activation was correlated with high Vimentin expression. In PDAC-, H&amp;N-, CRC-, and CK-low NICD4 cells, we observed an overexpression of NUMB, suggesting a correlation between low Notch4 activation and proteasomal degradation. In most tumor types, Notch4 activation was associated with an increased proliferation rate and basal migration. PF-03084014 had pronounced antiproliferative effects in high NICD4 cells in comparison to low NICD4 cells, except for HCC. In H&amp;N cells, PF-03084014 displayed no effect on Notch4 activation. We also observed a decrease in HES1 expression (significant in high NICD4 cells) and an increased in AKT phosphorylation (significant in low NICD4 cells). We will further analyze the effect of PF-03084014 on cell signaling and migration in other tumor types to display the results at the conference. Conclusions: In this study, we demonstrated a correlation between high Notch4 basal activation and increased proliferation/migration in a panel of human cancer cell lines, as well as higher sensitivity to the Notch inhibitor PF-03084014. In highly sensitive H&amp;N cell lines, PF-03084014 inhibited Notch signaling pathway, whereas we observed an activation of AKT survival pathway in the low-sensitive ones. Since Notch inhibition is an interesting topic for antitumor therapy, this study could help to select tumor types that could be good candidate for Notch inhibition in the clinic. Citation Format: Lucile Astorgues-Xerri, Matthieu Martinet, Eric Raymond, Sandrine Faivre, Annemilaï Tijeras-Raballand. Basal Notch4 activation is a druggable marker of aggressiveness in a panel of human cancer cell lines [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 859.

Trauma Induces Emergency Hematopoiesis through IL-1/MyD88-Dependent Production of G-CSF

Abstract The acute inflammatory response to infection or injury dramatically increases the hematopoietic demand on the bone marrow to replace effector leukocytes consumed in the inflammatory response. In the setting of infection, pathogen-associated molecular patterns induce emergency hematopoiesis, activating hematopoietic stem and progenitor cells to proliferate, thereby providing progeny for accelerated myelopoiesis. Sterile tissue injury due to trauma also increases leukocyte demand; however, the effect of sterile tissue injury on hematopoiesis is not well described. We used a mouse model of multisystem injury to investigate the effects of these injuries on bone marrow progenitor frequencies and phenotypes. We find that tissue injury alone induces emergency hematopoiesis in mice subjected to polytrauma. This process is driven by IL-1/MyD88-dependent production of G-CSF. G-CSF induces expansion of hematopoietic progenitors including hematopoietic stem cells and multipotent progenitors and increases the frequency of myeloid-skewed progenitors. These data provide the first comprehensive description of injury-induced emergency hematopoiesis and identify an IL-1/MyD88/G-CSF dependent pathway as the key regulator of emergency hematopoiesis after injury.

Early consequences of allopolyploidy alter floral evolution in Nicotiana (Solanaceae)

BackgroundPolyploidy has played a major role in angiosperm evolution. Previous studies have examined polyploid phenotypes in comparison to their extant progenitors, but not in context of predicted progenitor phenotypes at allopolyploid origin. In addition, differences in the trends of polyploid versus diploid evolution have not been investigated. We use ancestral character-state reconstructions to estimate progenitor phenotype at allopolyploid origin to determine patterns of polyploid evolution leading to morphology of the extant species. We also compare trends in diploid versus allopolyploid evolution to determine if polyploidy modifies floral evolutionary patterns.ResultsPredicting the ancestral phenotype of a nascent allopolyploid from reconstructions of diploid phenotypes at the time of polyploid formation generates different phenotype predictions than when extant diploid phenotypes are used, the outcome of which can alter conclusions about polyploid evolution; however, most analyses yield the same results. Using ancestral reconstructions of diploid floral phenotypes indicate that young polyploids evolve shorter, wider corolla tubes, but older polyploids and diploids do not show any detectable evolutionary trends. Lability of the traits examined (floral shape, corolla tube length, and corolla tube width) differs across young and older polyploids and diploids. Corolla length is more evolutionarily labile in older polyploids and diploids. Polyploids do not display unique suites of floral characters based on both morphological and color traits, but some suites of characters may be evolving together and seem to have arisen multiple times within Nicotiana, perhaps due to the influence of pollinators.ConclusionsYoung polyploids display different trends in floral evolution (shorter, wider corolla tubes, which may result in more generalist pollination) than older polyploids and diploids, suggesting that patterns of divergence are impacted by the early consequences of allopolyploidy, perhaps arising from genomic shock and/or subsequent genome stabilization associated with diploidization. Convergent evolution in floral morphology and color in Nicotiana can be consistent with pollinator preferences, suggesting that pollinators may have shaped floral evolution in Nicotiana.

Expression of Embryonic Stem Cell Markers in Microcystic Lymphatic Malformation.

Aim: To investigate the expression of embryonic stem cell (ESC) markers in microcystic lymphatic malformation (mLM). Methods and Results: Cervicofacial mLM tissue samples from nine patients underwent 3,3'-diaminobenzidine (DAB) immunohistochemical (IHC) staining for ESC markers octamer-binding protein 4 (OCT4), homeobox protein NANOG, sex determining region Y-box 2 (SOX2), Krupple-like factor (KLF4), and proto-oncogene c-MYC. Transcriptional activation of these ESC markers was investigated using real-time polymerase chain reaction (RT-qPCR) and colorimetric in situ hybridization (CISH) on four and five of these mLM tissue samples, respectively. Immunofluorescence (IF) IHC staining was performed on three of these mLM tissue samples to investigate localization of these ESC markers. DAB and IF IHC staining demonstrated the expression of OCT4, SOX2, NANOG, KLF4, and c-MYC on the endothelium of lesional vessels with abundant expression of c-MYC and SOX2, which was also present on the cells within the stroma, in all nine mLM tissue samples. RT-qPCR and CISH confirmed transcriptional activation of all these ESC markers investigated. Conclusions: These findings suggest the presence of a primitive population on the endothelium of lesional vessels and the surrounding stroma in mLM. The abundant expression of the progenitor-associated markers SOX2 and c-MYC suggests that the majority are of progenitor phenotype with a small number of ESC-like cells.

The epigenetic basis for the impaired ability of adult murine retinal pigment epithelium cells to regenerate retinal tissue

The epigenetic plasticity of amphibian retinal pigment epithelium (RPE) allows them to regenerate the entire retina, a trait known to be absent in mammals. In this study, we investigated the epigenetic plasticity of adult murine RPE to identify possible mechanisms that prevent mammalian RPE from regenerating retinal tissue. RPE were analyzed using microarray, ChIP-seq, and whole-genome bisulfite sequencing approaches. We found that the majority of key genes required for progenitor phenotypes were in a permissive chromatin state and unmethylated in RPE. We observed that the majority of non-photoreceptor genes had promoters in a repressive chromatin state, but these promoters were in unmethylated or low-methylated regions. Meanwhile, the majority of promoters for photoreceptor genes were found in a permissive chromatin state, but were highly-methylated. Methylome states of photoreceptor-related genes in adult RPE and embryonic retina (which mostly contain progenitors) were very similar. However, promoters of these genes were demethylated and activated during retinal development. Our data suggest that, epigenetically, adult murine RPE cells are a progenitor-like cell type. Most likely two mechanisms prevent adult RPE from reprogramming and differentiating into retinal neurons: 1) repressive chromatin in the promoter regions of non-photoreceptor retinal neuron genes; 2) highly-methylated promoters of photoreceptor-related genes.

CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability

One of the main challenges in limbal stem cell (LSC) biology and transplantation is the lack of definitive cell surface markers which can be used to identify and enrich viable LSCs. In this study, expression of 361 cell surface proteins was assessed in ex vivo expanded limbal epithelial cells. One marker, CD200 was selected for further characterization based on expression in a small subset of limbal epithelial cells (2.25% ± 0.69%) and reduced expression through consecutive passaging and calcium induced differentiation. CD200 was localized to a small population of cells at the basal layer of the human and mouse limbal epithelium. CD200+ cells were slow cycling and contained the majority of side population (SP) and all the holoclone forming progenitors. CD200+ cells displayed higher expression of LSCs markers including PAX6, WNT7A, CDH3, CK14, CK15, and ABCB5 and lower expression of Ki67 when compared to CD200- . Downregulation of CD200 abrogated the ability of limbal epithelial cells to form holoclones, suggesting an important function for CD200 in the maintenance and/or self-renewal of LSCs. A second marker, CD109, which was expressed in 56.29% ± 13.96% of limbal epithelial cells, was also found to co-localize with ΔNp63 in both human and mouse cornea, albeit more abundantly than CD200. CD109 expression decreased slowly through calcium induced cell differentiation and CD109+ cells were characterized by higher expression of Ki67, when compared to CD109- subpopulation. Together our data suggest that CD200 expression marks a quiescent population of LSCs with holoclone forming potential, while CD109 expression is associated with a proliferative progenitor phenotype. Stem Cells 2018;36:1723-1735.

Do Induced Pluripotent Stem Cell Characteristics Correlate with Efficient In Vitro Smooth Muscle Cell Differentiation? A Comparison of Three Patient-Derived Induced Pluripotent Stem Cell Lines.

Human induced pluripotent stem cells (iPSCs) have the potential to repair/regenerate smooth muscle cells (SMCs) in different organs. However, there are many challenges in their translation to clinical therapies. In this study, we describe our observations of in vitro SMC differentiation in three iPSC lines derived from human fibroblasts using retroviral, episomal, and mRNA/miRNA reprogramming methods. We sought to elucidate correlations between differentiation characteristics and efficiencies that can facilitate large-scale production of differentiated cells for clinical applications, and to report differences in pluripotency marker expression in differentiated cells from different iPSC lines. A standardized SMC differentiation protocol was used to induce the CD31+/CD34+ vascular progenitor cell phenotype. These were sorted by magnetic-activated (MACS) and fluorescence-activated cell sorting (FACS), and then treated with PDGF-BB and smooth muscle growth medium for further differentiation into smooth muscle progenitor cells (pSMCs). The expression of SMC and pluripotency markers in early- and late-passage (P1 and P4) pSMCs was analyzed. A total of 36 differentiation runs was performed on the three patient iPSC lines. All pSMC populations expressed SMC markers and Ki67 consistent with the progenitor phenotype. Initial iPSC density correlated positively with the sorted cell FACS efficiency, and this correlation could be fit to a quadratic equation. We also observed that a specific "honeycomb" pattern of the starting cultured iPSCs cultured correlated with higher efficiency in all three iPSC lines. Pluripotency marker expression decreased significantly to nearly undetectable levels in all three lines. There was no significant change in SMC and pluripotent marker expression between passage 1 and 4. In summary, our observations suggest that the method of iPSC reprogramming does not affect iPSC differentiation into pSMCs. Protocol efficiency can be modeled mathematically and coupled with the initial "honeycomb" cell pattern to optimize production of large cell numbers for clinical therapies.

Meso-Endothelial Bipotent Progenitors from Human Placenta Display Distinct Molecular and Cellular Identity.

SummaryThe existence of bipotential precursors for both mesenchymal and endothelial stem/progenitor cells in human postnatal life is debated. Here, we hypothesized that such progenitors are present within the human term placenta. From a heterogeneous placental single-cell suspension, a directly flow-sorted CD45−CD34+CD144+CD31Lo population uniquely differentiated into both endothelial and mesenchymal colonies in limiting dilution culture assays. Of interest, these bipotent cells were in vessel walls but not in contact with the circulation. RNA sequencing and functional analysis demonstrated that Notch signaling was a key driver for endothelial and bipotential progenitor function. In contrast, the formation of mesenchymal cells from the bipotential population was not affected by TGFβ receptor inhibition, a classical pathway for endothelial-mesenchymal transition. This study reveals a bipotent progenitor phenotype in the human placenta at the cellular and molecular levels, giving rise to endothelial and mesenchymal cells ex vivo.

Thymic B cell development is controlled by the B potential of progenitors via both hematopoietic-intrinsic and thymic microenvironment-intrinsic regulatory mechanisms.

BackgroundHematopoietic stem cells (HSCs) derived from birth through adult possess differing differentiation potential for T or B cell fate in the thymus; neonatal bone marrow (BM) cells also have a higher potential for B cell production in BM compared to adult HSCs. We hypothesized that this hematopoietic-intrinsic B potential might also regulate B cell development in the thymus during ontogeny.MethodsFoxn1lacZ mutant mice are a model in which down regulation of a thymic epithelial cell (TEC) specific transcription factor beginning one week postnatal causes a dramatic reduction of thymocytes production. In this study, we found that while T cells were decreased, the frequency of thymic B cells was greatly increased in these mutants in the perinatal period. We used this model to characterize the mechanisms in the thymus controlling B cell development.ResultsFoxn1lacZ mutants, T cell committed intrathymic progenitors (DN1a,b) were progressively reduced beginning one week after birth, while thymic B cells peaked at 3–4 weeks with pre-B-II progenitor phenotype, and originated in the thymus. Heterochronic chimeras showed that the capacity for thymic B cell production was due to a combination of higher B potential of neonatal HSCs, combined with a thymic microenvironment deficiency including reduction of DL4 and increase of IL-7 that promoted B cell fate.ConclusionOur findings indicate that the capacity and time course for thymic B-cell production are primarily controlled by the hematopoietic-intrinsic potential for B cells themselves during ontogeny, but that signals from TECs microenvironment also influence the frequency and differentiation potential of B cell development in the thymus.

Transcriptional profile of in vitro expanded human epidermal progenitor cells for the treatment of non-healing wounds

BackgroundEpidermal progenitor cells (EPCs) have been under extensive investigation due to their increasing potential of application in medicine and biotechnology. Cultured human EPCs are used in the treatment of chronic wounds and have recently became a target for gene therapy and toxicological studies. One of the challenges in EPCs culture is to provide a high number of undifferentiated, progenitor cells displaying high viability and significant biological activity. ObjectivesThe goal of this study was to characterize the in vitro cultured progenitor cells and to assess whether the cells with the progenitor phenotype are able to enhance wound healing. Additionally, we aimed to establish the complete procedure of the culture, analysis and clinical application of epidermal progenitor cells. MethodsIn this study we present a method of cell isolation and culture followed by a technique of transplantation of the cultured cells onto the wound bed. The applied isolation technique involves two enzymatic steps (dispase, trypsin) and it is characterized by a high yield of cells. The obtained cells were cultured in vitro up to the second passage in serum-free and xeno-free keratinocytes-dedicated medium. Key stem cell markers were determined with means of flow cytometry and quantitative real-time PCR. ResultsThe in vitro expanded cells displayed high proliferative activity without features of neither apoptosis nor necrosis. The flow cytometry and transcriptomic analyses showed enhanced expression of stem cell markers (i.e. proteins: ΔNp63, CD29, CD49f and BNC1, CDKN1A transcripts) in the expanded cells. In the presented compassionate use study, cultured autologous cells from an oncological patient were suspended in fibrin sealant and transplanted directly to a non-healing wound, resulting in wound closure within 2 months. ConclusionThe cells cultured in serum-free media display epidermal stem cells features and a potential to stimulate wound healing. This promising procedure of isolation, culture and application warrants further clinical trials in the treatment of chronic wounds.

Abstract 5078: EZH2 methyltransferase regulates disseminating tumor cells in breast cancer metastasis

Abstract Triple negative breast cancer (TNBC, ER-, PR-, HER2-) exhibits the worst outcome due to higher rates of metastasis compared to non TNBC subtypes. Despite this clinical significance, there is a conspicuous lack of FDA approved molecularly targeted anti-metastatic therapies for TNBC. The enhancer of zeste homolog 2 (EZH2), a catalytic core subunit of the Polycomb repressive complex 2 (PRC2) with histone methyltransferase (HMT) activity is associated with the worst clinical outcome in breast cancer patients. Using a combination of genetic and pharmacological approaches, we show that EZH2 HMT blockade did not impact primary tumor growth, but significantly reduced distal metastases. Metastasis suppression was associated with a marked reduction of tumor-initiating cells (TICs) in primary tumor, circulating tumor cells (CTCs) in the blood and impaired lung colonization. Using a SOX2/OCT4 promoter reporter system, we identified EZH2-sensitive metastatic cells with GATA3 low luminal progenitor phenotypes in the primary tumor, and EZH2 HMT blockade restored GATA3 expression, promoted differentiation of luminal progenitors and impaired metastasis. These key preliminary findings have led to the hypothesis that EZH2 promotes metastasis, and that inhibition of EZH2 HMT may constitute a viable anti-metastatic approach. We will also discuss the potential of EZH2 inhibition in combination with chemotherapy as an effective strategy against TNBC metastasis. Citation Format: Shira Yomtoubian, Seongho Ryu, Sharrell Lee, Geoff Markowitz, Dingcheng Gao, Vivek Mittal. EZH2 methyltransferase regulates disseminating tumor cells in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5078. doi:10.1158/1538-7445.AM2017-5078

Abstract LB-192: Phenotyping human myeloid derived suppressor cells (MDSC) subsets

Abstract MDSCs are heterogeneous occurring at varying stages of commitment and differentiation. They increase in cancer and other patients with inflammatory diseases and may best be defined based on their ability to suppress T and B cell responses. In recent years these cells have demonstrated clinical relevance, including a negative correlation with prognosis and overall survival in cancer patients increasing with tumor progression and stage. MDSCs were originally described in mice based on a lack of phenotypic markers, i.e. null cells, or predicated on their function, as natural suppressor (NS) cells. Subsequently they were found in humans, based on CD34 expression supporting a hematopoietic progenitor phenotype. The identification of human phenotype(s) is complicated by the association of suppressor cell function with multiple phenotypes. This includes two main subsets defined based on the expression or lack of myeloid and lymphoid markers, with specific phenotypes extent investigator dependent. There is a consensus that MDSCs constitute a continuum of immature to differentiated myeloid cells ranging from multi-potential hematopoietic progenitors to morphologically differentiated granulocytic and monocytic leukocytes. One trending hypothesis is that MDSC phenotypes form three subsets include macrophage (M-) MDSCs with a monocytic morphology, expressing CD14 and no CD15 or CD66b and granulocytic (G)-MDSCs phenotypically characterized by CD15 and/or CD66B expression and no CD14; and an immature (i-) MDSC defined as linage negative, CD33+ cells. Unfortunately, there is no consensus on markers extent to CD14 and CD15 although linage negative and DR- provide a common theme; although not commonly utilized between laboratories and MDSC subset. We provide herein a Flow cytometric strategy utilizing single staining tube and a linear phenotypic characterization that is supported by density and T-cell suppression confirmation. We suggest that staining with Lin, HLA-DR, CD14, CD15, CD11b, CD33 antibodies provides sufficient markers to subset into the three MDSC phenotypes that can be further subset with CD16. PDL1 and CD34 expression. Interestingly the majority of iMDSC express CD34 on the CD33+ cells together with CD14 or CD15 supporting a plastic phenotype with myeloid commitment. All subset can be further, delineated based on CD16 expression further suggesting immaturity. We will note, that some cancer patients express these cell phenotypes at very high levels in the circulation; although the preponderance of G-MDSC vs M-MDSC varies between patients. Citation Format: James E. Talmadge, Holly Britton, Alicia Dafferner, Phyllis Warkentin, Kathryn Cole. Phenotyping human myeloid derived suppressor cells (MDSC) subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-192. doi:10.1158/1538-7445.AM2017-LB-192

Phenotype and Function of Human Gut Hematopoietic Stem Cells and Progenitors

Lin−CD45+CD34+ cells have been reported in human small intestine and liver, but further phenotypic or functional analysis was not described[1][2]. We utilized flow cytometry and cytometry by time of flight (CyTOF) to study the composition and phenotype of human gut hematopoietic stem cells (HSCs) and hematopoietic progenitors (HPs) from deceased donors. Colony-forming-cell (CFC) assays, long-term-culture-initiating-cell (LTC-IC) assays and humanized mouse models were established to further evaluate the differentiation potential of gut HSCs/HPs. HSCs and multiple types of HPs, including multipotent progenitors (MPP), lymphoid-primed multipotent progenitors (LMPP), common lymphoid progenitors (CLP), myeloid progenitors (MP), and unclassified progenitors co-expressing CD56, were detected in human intestinal mucosa and mesenteric lymph nodes (MLNs). Ileum lamina propria lymphocytes (LPL) included significantly higher percentages of HSCs than intraepithelial lymphocytes (IEL). Both IEL and LPL showed significantly lower percentages of CLP compared to MLNs. From jejunum or ileum specimens of ITx patients collected from their stoma revision/closure (POD47- 1544), variable levels of Lin−CD45+CD34+ cells were detected (Figure 1A). However, when gating on HSCs, donor cells (>90%) predominated over recipient cells in four patients with chimerism, regardless of their ages or early clinical outcomes (Figure 1B-C). Lin−CD45+CD34+ cells from ileum LP and bone marrow (BM) demonstrated similar properties in viSNE plots from CyTOF analysis (Figure 2), suggesting similar functional phenotypes. Both ileum LP and BM Lin−CD45+CD34+ cells contained subpopulations expressing the thymus-homing marker CCR9, and T/NK lineage polarized progenitors coexpressing CD45RA and CD7. Additionally, CFC assays showed that both ileum LP and BM Lin−CD34-enriched cells can form myeloid/erythroid colonies, although ileum LP CD34 cells were less efficient than BM CD34 cells. LTC-IC assay using single cell sorted ileum LP HSC co-culture with murine feeder cells suggested myeloid/lymphoid differentiation potential of human gut HSCs. Experiments using human gut Lin−CD45+CD34+ cells to reconstitute humanized mice are ongoing.FIGURE 1: (A) Lin−CD45+CD34+ cells were detected in human intestinal mucosa long-term post-Tx (POD47-1544) (B) Donor-derived hematopoietic stemcells (HSCs) and/or multiple types of hematopoietic progenitors (HPs)were detected in transplanted small bowel specimens. (C) Composition of HSCs/HPs when gating on donor (upper panel) or recipient (lower panel) Lin−CD45+CD34+ cells.FIGURE 2: Lin−CD45+CD34+ cells from bone marrow (BM) and ileum lamina propria (LP) located in the same region of the viSNE plot from CyTOF analysis of individual organ donor, suggesting the similarity of their phenotype and origin.Taken together, our data suggest that human gut HSCs/HPs may be functionally similar to BM HSCs/HPs that have thymus-homing ability and can give rise to multiple lineages, which likely contribute to the long-persistent mixed chimerism post-ITx. Research reported in this publication was performed in the Columbia Center for Translational Immunology (CCTI) Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. CyTOF study was performed in collaboration with the Human Immune Monitoring Core (HIMC) of Icahn School of Medicine at Mount Sinai.

Human myeloid derived suppressor cell (MDSC) subset phenotypes

Abstract MDSCs are increased in cancer patients and were originally defined based on T-cell suppression and a negative correlation with prognosis and overall survival and a direct correlation with tumor stage. They were originally described, based on CD34 expression and a hematopoietic progenitor phenotype. However, their phenotypic definition is investigator dependent in association with suppressor cell function. There are two main subsets defined based on a lack of lymphoid and differential expression of myeloid markers resulting in a cellular continuum from multi-potential hematopoietic progenitors to differentiated granulocytes and monocytes. One recent hypothesis is that MDSCs form three subsets including macrophage MDSC (CD14+ and no CD15 or CD66b); granulocytic MDSCs (CD15 and/or CD66b expression and no CD14); and immature (i-) MDSC (linage (Lin) negative, CD33+ cells). Unfortunately, there is no consensus on markers extent to CD14 and CD15 with linage and HLA-DR negativity providing a frequent; although not universal theme. We report a flow cytometry gating strategy utilizing a single staining tube and a linear phenotypic characterization of MDSC subsets supported by density and T-cell suppression. We suggest that staining with antibodies to Lin, HLA-DR, CD14, CD15, CD11b and CD33 is sufficient to subset into the three phenotypes that can be further subset based on CD16. PDL1 and CD34 expression. The majority of iMDSC are CD34+CD33+ cells supporting a plastic phenotype and myeloid commitment and can be further, delineated based on CD16 expression supporting immaturity. We note, that some cancer patients express MDSCs at high levels; although the subsets preponderance varies between patients.

Endothelial Progenitors: A Consensus Statement on Nomenclature.

SummaryEndothelial progenitor cell (EPC) nomenclature remains ambiguous and there is a general lack of concordance in the stem cell field with many distinct cell subtypes continually grouped under the term “EPC.” It would be highly advantageous to agree on standards to confirm an endothelial progenitor phenotype and this should include detailed immunophenotyping, potency assays, and clear separation from hematopoietic angiogenic cells which are not endothelial progenitors. In this review, we seek to discourage the indiscriminate use of “EPCs,” and instead propose precise terminology based on defining cellular phenotype and function. Endothelial colony forming cells and myeloid angiogenic cells are examples of two distinct and well‐defined cell types that have been considered EPCs because they both promote vascular repair, albeit by completely different mechanisms of action. It is acknowledged that scientific nomenclature should be a dynamic process driven by technological and conceptual advances; ergo the ongoing “EPC” nomenclature ought not to be permanent and should become more precise in the light of strong scientific evidence. This is especially important as these cells become recognized for their role in vascular repair in health and disease and, in some cases, progress toward use in cell therapy. Stem Cells Translational Medicine 2017;6:1316–1320