Progenitor Phenotype Research Articles

The human chorion contains definitive hematopoietic stem cells from the fifteenth week of gestation.

We examined the contribution of the fetal membranes, amnion and chorion, to human embryonic and fetal hematopoiesis. A population of cells displaying a hematopoietic progenitor phenotype (CD34++ CD45low) of fetal origin was present in the chorion at all gestational ages, associated with stromal cells or near blood vessels, but was absent in the amnion. Prior to 15 weeks of gestation, these cells lacked hematopoietic in vivo engraftment potential. Differences in the chemokine receptor and β1 integrin expression profiles of progenitors between the first and second trimesters suggest that these cells had gestationally regulated responses to homing signals and/or adhesion mechanisms that influenced their ability to colonize the stem cell niche. Definitive hematopoietic stem cells, capable of multilineage and long-term reconstitution when transplanted in immunodeficient mice, were present in the chorion from 15-24 weeks gestation, but were absent at term. The second trimester cells also engrafted secondary recipients in serial transplantation experiments. Thus, the human chorion contains functionally mature hematopoietic stem cells at mid-gestation.

TLR Activation By MSC Derived Extracellular Vesicles Drives Myeloid Commitment

Hematopoietic stem and progenitor cells (HSPC) are responsive to paracrine signaling in the bone marrow microenvironment. We recently showed that leukemia derived extracellular vesicles (EVs) directly regulate HSPC in the leukemic niche and confer a net loss of progenitor function in part through direct action of a highly abundant miRNA on the hematopoietic transcription factor c-Myb. These observations motivated us to explore the potential regulation of HSPC by EVs present during steady state hematopoiesis in the unperturbed bone marrow. Mesenchymal stromal cells (MSC) comprise a diverse set of cells secreting supportive factors central to the maintenance of HSPC. We systematically characterized EVs released by murine MSCs as a candidate population and demonstrated that EVs themselves carry cytokines, predominantly interleukin (IL) -1, -2, -4,-6 and -17A, as well as G-CSF and Interferon g. As a functional corollary, we found that MSC EVs led to preferential granulocyte and monocyte differentiation in co-culture with C-kit enriched HSPCs, when compared to vesicle free condition (termed S100). These observations were further supported by the expansion of KSL (c-kit+,Sca-1+,lin-) and MPP2-4 (CD48+, CD150+/-) progenitor populations. To determine the in vivo relevance of these findings we performed a series of competitive transplantation experiments, that demonstrated that MSC-EV exposed HSPCs outperformed HSPCs cultured with EV depleted media, with striking advantage in early, but a significant deficit in later (week 16) myeloid recovery, compared to the S100 condition. These observations were broadly consistent with recent evidence for HSPC regulation by toll like receptors (TLRs) and we confirmed that the observed deficit in progenitor phenotypes was partially reversed in MSC-EV co-cultures with HSPCs from animals with genetic disruption of TLR4 or MyD88. EV-TLR engagement further induced a NF-kB response with downstream transcriptional activation of canonical transcriptional targets (STAT1, IRF1, -9) and significantly upregulated the release of IL-3, -6, IFNg, and G-CSF in exposed WT HSPC, compared with S100. Finally, we performed deconvolution microscopy studies of co-cultured HSPC and demonstrated the Pearson's spatial correlation between fluorescently tagged MSC-EV and TLR4 as well as the early and late endosomal marker proteins, Rab5 and Rab7, respectively. We conclude that MSC-derived EV trafficking of TLR contributes to the homeostatic regulation of HSPCs in the bone marrow microenvironment as a mechanism of tonic regulation of hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Src-like Adaptor Protein Promotes the CD103+ dendritic Cell Homeostasis in the Lung

Dendritic cells (DCs) along with mast cells function as sentinels for the innate immune system and perform as antigen presenting cells (APCs) to mount an adaptive immune response against invading pathogen. FLT3 receptor tyrosine kinase signaling has been shown to regulate the homeostatic mechanisms of subsets of DCs particularly, CD103+DCs compared to CD11b+DCs. CD103+DCs are regarded as APCs with superior capabilities to mount an effective immune response, thus understanding their homeostasis mechanism(s)/function is of paramount importance to devise effective therapeutics including DC vaccines. The Src-like adapter protein (SLAP) has been shown to dampen the signaling downstream of receptor tyrosine kinases including FLT3, cKit, and immune cell receptors including T cell receptor, B cell receptor, and Granulocyte-monocyte colony stimulating factor receptor via by recruiting c-Cbl, an ubiquitin ligase.Here, we report that SLAP deficient mice (KO) have reduced numbers of CD103+DC in lung while equal numbers in liver and kidney compared to control mice. To further confirm reduced CD103+DC in the lung, efferocytosis assays that are dependent upon CD+103 DC in lung epithelium to cleanse the apoptotic cells were performed. Flow cytometric quantification of CD103+DCs that uptake fluorescently labeled apoptotic cells administered via intranasal route and migrate to mediastinal lymph nodes confirmed reduced number of CD103+DCs in SLAP KO mice. Further analysis of DC progenitor populations showed reduced pre-DC progenitor in the lung in SLAP KO mice while bone marrow compartment showed equal progenitor populations including pre-DC and common dendritic progenitors suggesting the role of SLAP in localized FLT3 signaling in the lung. Consistently, DCs in lymphoid compartment including spleen, thymus, inguinal and popliteal lymph node did not show any defects. Upon further dissecting the cellular mechanism, SLAP KO DCs showed increased apoptosis while having similar proliferation potential in vivo at steady state.Bone marrow progenitors from SLAP KO mice failed to generate mature DCs in the presence of FLT3 ligand in vitrodue to enhanced apoptosis at early time points. Also, submaximal inhibition of FLT3 with an inhibitor, quizartinib partially rescues the apoptotic phenotype of SLAP KO bone marrow progenitors suggesting a cell-intrinsic role of SLAP in the survival of DCs. Biochemical analysis revealed that SLAP is directly recruited to the juxta-membrane residues of the FLT3 receptor in an inducible manner suggesting a direct role of SLAP in the regulation of FLT3 signaling. Phosphoflow analysis of DCs generated in the combined presence of GMCSF and FLT3 ligands showed that SLAP promotes the signaling to SHP2 while perturbs signaling to the mTOR pathway. Together these results suggest that SLAP is a critical regulator of CD103+DCs homeostasis in selective peripheral organs including the lung. DisclosuresNo relevant conflicts of interest to declare.

Spatiotemporal Mapping of Erythroid, Stromal, and Osteogenic Niche Formation to Support Physiologic Red Cell Production in a Three-Dimensional Hollow Fibre Perfusion Bioreactor

Current in vitro human erythroid culture platforms require abnormally high cytokine supplementation and use lower cell density (<106/mL) compared with that present in bone marrow during physiologic erythropoiesis. These in vitro culture conditions limit extracellular interactions, are not dynamic, and exhaust stem and progenitor cell pools, thereby limiting culture longevity. We have developed a three-dimensional hollow fibre perfusion bioreactor (HFBR) comprised of a collagen-coated polyurethane scaffold which surrounded ceramic hollow fibres (HFs) and expanded dense inocula of human umbilical cord blood (CB) mononuclear cells (MNCs; >107/mL) when perfused with cytokine-free media in long-term culture. In order to study the role of this manufactured HFBR microenvironment on spatiotemporal physiologic erythropoiesis, we now extend our previous reports by implementing 5-fold less cytokine concentrations than those used in typical ex vivo erythropoietic cultures. Herein, we show a >107 red cell harvest from the HFBR culture over 28-days with spontaneous expansion of stromal cells, maintenance of erythroid progenitor pools, and formation of stromal and erythroid cell niches in defined areas within the HFBR structure with differential in situ production of 23 growth factors varying over time.The 5.25 mL HFBR scaffold was inoculated with 108 CBMNCs and HFs were rapidly perfused (20 mL/h) with serum-free StemSpan medium gradually supplemented with a cytokine gradient of decreasing SCF (50 - 0 ng/mL) and increasing EPO (0 - 0.3 U/mL) over 28 days in order to maintain progenitor cells whilst inducing erythropoiesis. Quantitative confocal microscopy analyses of HFBR sections demonstrated that DAPI+ CBMNCs maintained high cell density (>107/mL), and high viability (>80%), while more than 107 enucleated cells were filtered through HFs over the 28-day culture. Inside the HFBR, hematopoietic progenitor cells were maintained (total of 3.1·106 CD34+ and 5.5·106 CKIT+ MNCs) while erythroid cells were expanded across various stages of maturation (28-day total increase of 1.2·107 EPOR+, 1.8·107 CD71+, and 2.3·107 CD235a+ MNCs); CD235a+mature red cell phenotypes were enriched 10-fold in the HF filtrate over 28 days.Stromal cells expanded and differentiated during the 28-day HFBR culture with a total increase of mesenchymal stem cell marker Stro-1 (2.2·107 cells), pre-osteoblast marker osterix (OSx; 1.6·107 cells), and mature osteoblast marker osteopontin (OPN; 0.5·107 cells). Expression of human collagen-1, fibronectin, and laminin-2 was detected by microscopy, while enzyme-linked immunoassays on HFBR filtrate detected 23 multilineal, unsupplemented cytokine profiles including interleukins produced primarily from day 0-12 (IL-6, IL-10, IL-21) as well as colony stimulating factors and stromal growth factors which increased in production from day 20-28 (G-CSF, GM-CSF, EGF, VEGF, Ang-2, PDGF, FGF-β). Using a novel confocal microscopy computational analyses that we have developed, DAPI+MNCs were found to self-associate into expanding 50-500µm clusters throughout the 28-day culture which increased local cell density 10-20 fold, representing niche-like areas. At day 14 and 28, MNCs formed clustered niches far from HFs which expressed hypoxic (HIF1a, PIMO), stromal, and erythroid markers (Stro-1, OSx, collagen-1, laminin-2, VCAM-1, CD45, EPOR: >1400µm from HFs). At day 28, 3-fold more MNC clusters formed near HFs and were comprised of hematopoietic progenitor and erythroid phenotypes (CD45, CD34, CKIT, CD235a, CD71: <700µm from HFs).Our data suggested that the dense inoculation of CBMNCs in a serum-free HFBR platform using physiologic concentrations of SCF and EPO enabled the long-term simultaneous differentiation of human erythroid, stromal, and osteogenic lineages, and the generation of an ex vivo erythroid inductive environment. This environment maintained multilineal progenitors, enabled harvest of mature erythrocytes, generated cytokine support in situ, and formed interactive cell niches which could be quantitatively mapped in spatiotemporal zones. The HFBR we have developed may represent a more physiologically-relevant culture system to study ex vivo erythropoiesis and could potentially provide a platform for translational cell expansion protocols. DisclosuresNo relevant conflicts of interest to declare.

The Mechanism By Which Mutant Nucleophosmin (NPM1) Creates Leukemic Self-Renewal Is Readily Reversed

Acute myeloid leukemia (AML) is self-renewal by immature myeloid precursors that fail to differentiate. An influential 'leukemia stem cell' model thus proposes that leukemogenic proteins augment or introduce a stem cell-like self-renewal program into cells, e.g., by upregulating signaling or transcription factors (TF) emblematic of stem cells (e.g., HOX). We investigated how the most recurrently mutated protein in AML, mutant nucleophosmin (mNPM1), causes leukemic cell expansion. The results challenge this model, but most importantly, open the door to rational targeted therapy for mNPM1 AML. One way of examining for stem cell programs in AML cells is to look at expression patterns of master TF that regulate expression of hundreds of genes and dictate cell fates. Of these select TF, the master TF that create hematopoietic stem cells (HLF etc.) are minimally or not expressed. Instead, there are very high levels of the master TF that drive monocyte and granulocyte lineage fates, PU.1 (SPI1) and CEBPA. Clearly, however, the lineage-programs intended by PU.1/CEBPA are inefficiently executed if at all - mNPM1 AML patient bone marrows had 85-97% cells with a granulocyte-monocyte progenitor phenotype, accumulated at the expense of downstream mature cells (Quek et al, JEM 2016). This aggregation at a lineage-committed, intermediate, naturally proliferative level of the hematopoietic hierarchy suggests an alternative model - instead of introducing a poorly-defined stem cell self-renewal program, mutant proteins disable differentiation programs which usually quench MYC-driven proliferation intrinsic to lineage-progenitors. To better understand how mNPM1 interacts with cellular machinery, we used mass-spectrometry to comprehensively document the protein interactions of endogenous NPM1 in AML cell nuclear and cytoplasmic fractions, the first analysis of this kind. Notably, the NPM1 protein interactome was enriched for PU.1. Critically, wild-type (wt) NPM1/PU.1 was in the nucleus of wtNPM1 AML cells, but mNPM1/PU.1 was in the cytoplasm of mNPM1 AML cells. This was evident clearly also by Western blot of cell fractions and by IF microscopy of primary AML cells and cell lines. Is cytoplasmic dis-location of PU.1 sufficient to explain persistent hematopoietic precursor proliferation? We used murine Pu.1 knock-out hematopoietic precursors transduced to express Pu.1 fused with the estrogen receptor (Pu.1-ER) to answer this question - Pu.1 relocation from the cytoplasm to the nucleus by tamoxifen triggered monocytic differentiation that terminated proliferation. Moreover, Pu.1-ER cells, like mNPM1 AML cells, highly express Hox genes, rapidly suppressed upon Pu.1 relocation to the nucleus. Thus, Pu.1 dominantly controls Hox and proliferation, as befitting of a master TF, and persistent HOX expression, like persistent progenitor proliferation, can be caused by Pu.1 loss-of-function. Protein macromolecules like NPM1 require transport factors to exit (exportins) the nucleus. A specific exportin, XPO1, was the major exportin found in the NPM1 interactome. XPO1 interactions with transported cargo can be inhibited by the small molecule drug KPT330. KPT330 10-20 nM rapidly re-located mNPM1 and PU.1 to the nucleus, downregulated MYC, upregulated p27/CDKN1B, upregulated monocyte but not granulocyte differentiation markers, induced morphologic changes of monocyte differentiation, and terminated proliferation of mNPM1 AML cells. The same low nanomolar treatment did not induce differentiation of wtNPM1 AML cells (THP1). Moreover, these KPT330 levels are not toxic to normal hematopoiesis (also shown by others). Thus, rather than gain-of-function of elusive stem cell-like self-renewal, the most frequently mutated protein in AML creates self-renewal by disabling a differentiation program that quenches intrinsic MYC-driven proliferation of lineage-progenitors. These observations are a mechanistic rationale to select refractory/relapsed mNPM1 AML patients for treatment with low well-tolerated doses of KPT330, with a defined molecular pharmacodynamic objective of returning PU.1 to the nucleus, to produce cell cycle exits by differentiation rather than p53-mediated apoptosis (to address chemotherapy resistance), to spare precious normal HSC (good therapeutic index), and directly reverse the basis for leukemic self-renewal (proliferation without differentiation). [Display omitted] DisclosuresLandesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder. Saunthararajah:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine.

Phenotypic Screen for Cardiac Regeneration Identifies Molecules with Differential Activity in Human Epicardium-Derived Cells versus Cardiac Fibroblasts.

Activation and proliferation of resident cardiac progenitor cells has therapeutic potential to repair the heart after injury. However, research has been impeded by a lack of well-defined and characterized cell sources and difficulties in translation to screening platforms. Here, we describe the development, validation, and use of a 384-well phenotypic assay in primary human epicardium-derived cells (EPDCs) to identify compounds that induce proliferation while maintaining the progenitor phenotype. Using this assay, we screened 7400 structurally diverse compounds where greater than 90% are biologically annotated and known to modulate a broad range of biological targets. From the primary screen, we identified and validated hits and expanded upon the lead molecules of interest. A counterscreen was developed in human cardiac fibroblasts to filter out compounds with a general proliferative effect, after which the activity of selected molecules was confirmed across multiple EPDC donors. To further examine the mechanism of action of compounds with annotated targets, we performed knockdown experiments to understand whether a single known target was responsible for the proliferative effect, confirming results with protein expression and activity assays. Here, we were able to show that the annotated targets of compounds of interest were not responsible for the proliferative effect, which highlights potential differences in cell types and signaling pathways and possible polypharmacology. These studies demonstrate the feasibility of using relevant human primary cells in a phenotypic screen to identify compounds as novel biological tools and starting points for drug discovery projects, and we disclose the first small molecules to proliferate human primary EPDCs.

Endogenous spinal cord stem cells in multiple sclerosis and its animal model

The adult mammalian spinal cord (SC) harbors neural stem cells (NSCs). The SC-NSCs are mostly quiescent during physiological conditions but are quickly activated in traumatic injury models. The SC-NSCs generate mostly glia, but are able to differentiate into neurons when affected by favourable conditions. An example is the inflammatory milieu in the SC of rat EAE, where the SC-NSCs migrate into demyelinated lesions and give rise to both glia and neurons. In MS, cells with progenitor phenotypes accumulate in inflammatory lesions both in brain and SC, but the extent to which these cells contribute to repair remains to be revealed.

Oct4 Methylation-Mediated Silencing As an Epigenetic Barrier Preventing Müller Glia Dedifferentiation in a Murine Model of Retinal Injury.

Müller glia (MG) is the most abundant glial type in the vertebrate retina. Among its many functions, it is capable of responding to injury by dedifferentiating, proliferating, and differentiating into every cell types lost to damage. This regenerative ability is notoriously absent in mammals. We have previously reported that cultured mammalian MG undergoes a partial dedifferentiation, but fails to fully acquire a progenitor phenotype and differentiate into neurons. This might be explained by a mnemonic mechanism comprised by epigenetic traits, such as DNA methylation. To achieve a better understanding of this epigenetic memory, we studied the expression of pluripotency-associated genes, such as Oct4, Nanog, and Lin28, which have been reported as necessary for regeneration in fish, at early times after NMDA-induced retinal injury in a mouse experimental model. We found that although Oct4 is expressed rapidly after damage (4 hpi), it is silenced at 24 hpi. This correlates with a significant decrease in the DNA methyltransferase Dnmt3b expression, which returns to basal levels at 24 hpi. By MS-PCR, we observed a decrease in Oct4 methylation levels at 4 and 12 hpi, before returning to a fully methylated state at 24 hpi. To demonstrate that these changes are restricted to MG, we separated these cells using a GLAST antibody coupled with magnetic beads. Finally, intravitreous administration of the DNA-methyltransferase inhibitor SGI-1027 induced Oct4 expression at 24 hpi in MG. Our results suggest that mammalian MG injury-induced dedifferentiation could be restricted by DNA methylation, which rapidly silences Oct4 expression, preventing multipotency acquisition.

Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by Klf4.

The vascular adventitia is a complex layer of the vessel wall consisting of vasa vasorum microvessels, nerves, fibroblasts, immune cells, and resident progenitor cells. Adventitial progenitors express the stem cell markers, Sca1 and CD34 (adventitial sca1-positive progenitor cells [AdvSca1]), have the potential to differentiate in vitro into multiple lineages, and potentially contribute to intimal lesions in vivo. Although emerging data support the existence of AdvSca1 cells, the goal of this study was to determine their origin, degree of multipotency and heterogeneity, and contribution to vessel remodeling. Using 2 in vivo fate-mapping approaches combined with a smooth muscle cell (SMC) epigenetic lineage mark, we report that a subpopulation of AdvSca1 cells is generated in situ from differentiated SMCs. Our data establish that the vascular adventitia contains phenotypically distinct subpopulations of progenitor cells expressing SMC, myeloid, and hematopoietic progenitor-like properties and that differentiated SMCs are a source to varying degrees of each subpopulation. SMC-derived AdvSca1 cells exhibit a multipotent phenotype capable of differentiating in vivo into mature SMCs, resident macrophages, and endothelial-like cells. After vascular injury, SMC-derived AdvSca1 cells expand in number and are major contributors to adventitial remodeling. Induction of the transcription factor Klf4 in differentiated SMCs is essential for SMC reprogramming in vivo, whereas in vitro approaches demonstrate that Klf4 is essential for the maintenance of the AdvSca1 progenitor phenotype. We propose that generation of resident vascular progenitor cells from differentiated SMCs is a normal physiological process that contributes to the vascular stem cell pool and plays important roles in arterial homeostasis and disease.

Single Cell Clones Purified from Human Parotid Glands Display Features of Multipotent Epitheliomesenchymal Stem Cells.

A better understanding of the biology of tissue-resident stem cell populations is essential to development of therapeutic strategies for regeneration of damaged tissue. Here, we describe the isolation of glandular stem cells (GSCs) from a small biopsy specimen from human parotid glands. Single colony-forming unit-derived clonal cells were isolated through a modified subfractionation culture method, and their stem cell properties were examined. The isolated clonal cells exhibited both epithelial and mesenchymal stem cell (MSC)-like features, including differentiation potential and marker expression. The cells transiently displayed salivary progenitor phenotypes during salivary epithelial differentiation, suggesting that they may be putative multipotent GSCs rather than progenitor cells. Both epithelial and mesenchymal-expressing putative GSCs, LGR5+CD90+ cells, were found in vivo, mostly in inter-secretory units of human salivary glands. Following in vivo transplantation into irradiated salivary glands of mice, these cells were found to be engrafted around the secretory complexes, where they contributed to restoration of radiation-induced salivary hypofunction. These results showed that multipotent epitheliomesenchymal GSCs are present in glandular mesenchyme, and that isolation of homogenous GSC clones from human salivary glands may promote the precise understanding of biological function of bona fide GSCs, enabling their therapeutic application for salivary gland regeneration.

P53 Regulates Progenitor Cell Quiescence and Differentiation in the Airway

Mechanisms that regulate progenitor cell quiescence and differentiation in slowly replacing tissues are not fully understood. Here, we demonstrate that the tumor suppressor p53 regulates both proliferation and differentiation of progenitors in the airway epithelium. p53 loss decreased ciliated cell differentiation and increased the self-renewal and proliferative capacity of club progenitors, increasing epithelial cell density. p53-deficient progenitors generated a pseudostratified epithelium containing basal-like cells invitro and putative bronchioalveolar stem cells invivo. Conversely, an additional copy of p53 increased quiescence and ciliated cell differentiation, highlighting the importance of tight regulation of p53 levels. Using single-cell RNA sequencing, we found that loss of p53 altered the molecular phenotype of progenitors and differentially modulated cell-cycle regulatory genes. Together, these findings reveal that p53 is an essential regulator of progenitor cell behavior, which shapes our understanding of stem cell quiescence during homeostasis and in cancer development.

Phenotypic Analysis of Stromal Vascular Fraction after Mechanical Shear Reveals Stress-Induced Progenitor Populations.

Optimization of fat grafting continues to gain increasing attention in the field of regenerative medicine. "Nanofat grafting" implements mechanical emulsification and injection of standard lipoaspirate for the correction of superficial rhytides and skin discoloration; however, little is known about the cellular constituents of the graft. Based on recent evidence that various stressors can induce progenitor activity, the authors hypothesized that the shear forces used in common fat grafting techniques may impact their regenerative capacities. Lipoaspirates were obtained from 10 patients undergoing elective procedures. Half of each sample was subjected to nanofat processing; the other half was left unchallenged. The viscosity of each sample was measured for computational analysis. The stromal vascular fraction of each sample was isolated, quantified, and analyzed by means of flow cytometry with two multicolor fluorescence antibody panels. Standard lipoaspirate is ideally suited for mechanical stress induction. The mechanical emulsification involved in nanofat processing did not affect cell number; however, viability was greatly reduced when compared with the stromal vascular fraction of standard lipoaspirate. Interestingly, nanofat processing resulted in stress-induced stromal vascular fraction with a higher proportion of endothelial progenitor cells, mesenchymal stem cells, and multilineage differentiating stress-enduring cells. Single-parameter analysis also revealed significant increases in CD34, CD13, CD73, and CD146 of the stress-induced stromal vascular fraction, markers associated with mesenchymal stem cell activity. Mechanical processing used in techniques such as nanofat grafting induces the up-regulation of progenitor phenotypes consistent with multipotency and pluripotency. These data provide a first step in characterizing the potential regenerative benefits realized through stress induction in fat grafting. Therapeutic, V.

Partial Conservation between Mice and Humans in Olfactory Bulb Interneuron Transcription Factor Codes.

The mammalian main olfactory bulb (OB) has a large population of GABAergic inhibitory interneurons that contains several subtypes defined by the co-expression other neurotransmitters and calcium binding proteins. The three most commonly studied OB interneuron subtypes co-express either Calretinin, Calbindin, or Tyrosine hydroxylase (Th). Combinations of transcription factors used to specify the phenotype of progenitors are referred to as transcription factor codes, and the current understanding of transcription factor codes that specify OB inhibitory neuron phenotypes are largely based on studies in mice. The conservation of these transcription factor codes in the human OB, however, has not been investigated. The aim of this study was to establish whether transcription factor codes in OB interneurons are conserved between mice and humans. This study compared the co-expression of Foxp2, Meis2, Pax6, and Sp8 transcription factors with Calretinin, Calbindin, or Th in human and mouse OB interneurons. This analysis found strong conservation of Calretinin co-expression with Sp8 and Meis2 as well as Th co-expression with Pax6 and Meis2. This analysis also showed that selective Foxp2 co-expression with Calbindin was conserved between mice and humans, which suggests Foxp2 is a novel determinant of the OB Calbindin interneuron phenotype. Together, the findings in this study provide insight into the conservation of transcription codes for OB interneuron phenotypes between humans and mice, as well as reveal some important differences between the species. This advance in our understanding of transcription factor codes in OB interneurons provides an important complement to the codes that have been established for other regions within the mammalian central nervous system, such as the cortex and spinal cord.

Abstract 1175: Lossof claudin-3 expression induces de-differentiation in colonic epithelial cellsto promote colon cancer malignancy and associates with poor patient survival

Abstract Dysregulation of colonocyte differentiation, imposing a crypt progenitor phenotype, characterize colorectal cancer (CRC) progression. Thus, an improved understanding of the molecular processes that regulate colonocyte differentiation can help identify novel therapeutic targets and biomarkers. In this regard, we found claudin-3, a tight junction integral protein, to be the highest expressed cell-cell adhesion moiety in the normal colonic epithelium and particularly concentrated amongst terminally differentiated colonocytes at the crypt top. We therefore postulated a key role for claudin-3 in maintaining colonocyte differentiation and negative association with colon tumorigenesis. In accordance, claudin-3 expression was low in 10 out of 14 CRC cells lines tested and undetectable in poorly differentiated and highly tumorigenic HCT116 and SW620 cell lines. In further analysis, using mRNA and protein expression, and utilizing samples from a large patient cohort (&amp;lt;250 CRC specimen), we found claudin-3 expression to be significantly suppressed (p&amp;lt;0.001 versus normal) in cancer tissues versus normal mucosa. The colon tissues from the established mouse models of colon cancer (APCmin mice and Azoxymethane (AOM)-DSS-induced colon cancer) demonstrated similar tumor specific decrease in claudin-3 expression. Interestingly, claudin-3 negative tumors retained E-cadherin expression. Most notably, we found a significant and positive correlation (p&amp;lt;0.001) of the greater levels of claudin-3 expression with patient survival. Genetic manipulation studies using colon cancer cells further supported a causal role of claudin-3 in upholding colonocyte differentiation, by modulating ZEB-1 protein synthesis and Wnt-signaling activation (Topflash promoter activity, p&amp;lt;0.05; claudin-3 siRNA versus control cells), and inhibiting cancer cell mobility and invasive ability. Importantly, homozygous deletion of claudin-3 expression in mice similarly inhibited colonocyte differentiation (down-regulated P-27 expression and up-regulated Vimentin expression), and promoted colon cancer growth and invasion when subjected to the AOM-DSS-induced mouse model of colorectal cancer (p&amp;lt;0.001 versus WT littermates). Pharmacological manipulations further revealed epigenetic regulation as potential mechanism for the inhibition of caludin-3 expression in CRC. Taken together, our data reveal a tissue-specific and inverse causal association of claudin-3 expression with CRC progression and patient survival, and highlights key importance of claudin-3 in maintaining colonocyte differentiation in Wnt-ZEB1-dependent manner. Citation Format: Rizwan Ahmad, Zhimin Chen, Balawant Kumar, Xi Chen, Mary kay Washington, Punita Dhawan, Amar B. Singh. Lossof claudin-3 expression induces de-differentiation in colonic epithelial cellsto promote colon cancer malignancy and associates with poor patient survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1175.

Finding Expandable Induced Cardiovascular Progenitor Cells.

Expandable Cardiovascular Progenitor Cells Reprogrammed From Fibroblasts Zhang et al Cell Stem Cell . 2016;18:368–381. Lineage Reprogramming of Fibroblasts Into Proliferative Induced Cardiac Progenitor Cells by Defined Factors Lalit et al Cell Stem Cell . 2016;18:354–367. Cardiovascular progenitor cells (CPCs) are a promising cell source for cardiac regenerative therapy because of their ability for self-renewal and differentiation into various cardiovascular cell types beneficial for myocardial repair: cardiomyocytes, smooth muscle cells, and endothelial cells. Previous evaluations of exogenously derived CPCs have focused mainly on their capacity for trilineage differentiation rather than self-renewal because of the lack of an effective protocol to maintain and expand CPCs long-term in culture. In a recent issue of Cell Stem Cell , 2 groups of investigators independently reported their success in isolating, maintaining, and expanding mouse CPCs in culture for >18 to 20 passages (1010- to 1015-fold expansion), with faithful preservation of progenitor phenotype and ability for trilineage-restricted differentiation in both cell culture and mouse models of myocardial infarction (MI). This commentary will discuss the unique findings of these 2 studies, highlight the strengths and weaknesses of each CPC derivation/expansion technique, and propose additional steps necessary to accelerate their clinical translation. The past 2 decades have witnessed some of the most dramatic developments in the field of cardiac regeneration. Central to numerous controversies is the debate on whether certain stem cell types can differentiate into functional cardiomyocytes at a clinically relevant efficiency after implantation for repair of MI. Bone marrow–derived hematopoietic stem cells1,2 and c-kit+ resident cardiac stem cells3 are 2 notable cell types at the center of contention. Although many adult stem cells can still confer modest therapeutic benefits via paracrine effects, the search for a bona fide progenitor cell type that can generate a significant …

Secreted HMGB1 from Wnt activated intestinal cells is required to maintain a crypt progenitor phenotype.

Background and AimsColorectal cancer (CRC) arises via multiple genetic changes. Mutation of the tumour suppressor gene APC, a key regulator of Wnt signalling, is recognised as a frequent early driving mutation in CRC. We have previously shown that conditional loss of Apc within the murine small intestine (Apcfloxmice) results in acute Wnt signalling activation, altered crypt-villus architecture and many hallmarks of neoplasia. Our transctipomic profiling (Affymetrix Microarrays) and proteomic profiling (iTRAQ-QSTAR) of Apc-deficient intestine inferred the involvement of High Mobility Group Box 1 (Hmgb1) in CRC pathogenesis. Here we assess the contribution of HMGB1 to the crypt progenitor phenotype seen following Apc loss.ResultsElevated HMGB1 was confirmed in intestinal epithelia and serum following conditional loss of Apc. Treatment of Apcflox mice with anti-HMGB1 neutralising antibody significantly reduced many of the crypt progenitor phenotypes associated with Apc loss; proliferation and apoptosis levels were reduced, cell differentiation was restored and the expansion of stem cell marker expression was eradicated.MethodsHmgb1 levels in intestinal epithelia and serum in Apcflox and ApcMin mice were assessed using qRT-PCR, Western blot and ELISA assays. The functional importance of elevated extracellular Hmgb1 was assessed using an anti-HMGB1 neutralising antibody in Apcflox mice.ConclusionsHMGB1 is expressed and secreted from intestinal epithelial cells in response to Wnt signalling activation. This secreted HMGB1 is required to maintain nearly all aspects of the crypt progenitor phenotype observed following Apc loss and add to the body of accumulating evidence indicating that targeting HMGB1 may be a viable novel therapeutic approach.

Thymic epithelial cells control let-7 up-regulation in thymic B progenitors during the neonatal to adult transition

Abstract The postnatal thymus is an efficient microenvironment for T cell specification and differentiation. B cells are also present in the thymus, and have been recently implicated in the central tolerance. In Foxn1lacZmutant mice, which undergo premature thymic involution beginning 1 week after birth, T committed progenitors were progressively reduced, however, thymic B cells started to increase at 1 week and transiently formed a peak at 3–4 weeks. These increased B cells were developed from neonatal derived BM progenitors possessing a high B potential, were originally generated in the thymus. Most of them showed CD19loB220loCD24hiCD43+/−IgM−progenitor phenotype with increased expression of Ly51 but decrease of CD25 accumulating at pre-B-II stage. These B progenitors showed a delayed down-regulation of Lin28b, an impaired up-regulation of Let-7 with an increased expression of Arid3a. However the treatment of these progenitor B cells with Vitamin D3 might up-regulate Let-7 and down-regulate Arid3 in vivo coculture. These changes were associated with a transient increase in IL7, decreased CCL25, DL4 and Cyp27b1, IL15 in thymic epithelial cells (TECs), which also reflected the changes of progenitor switch from fetal to adult type during neonatal. Our data show that signaling provided by TECs control the development of thymic B cells by regulating Let-7 and Arid3a expressions in the thymic progenitor B cells during the neonatal to adult transition.

558. Targeted Gene Therapy in CD34+ Cells from Healthy Donors and Fanconi Anemia Patients

Gene targeting is rapidly progressing thanks to the development of improved nucleases and donor constructs, and represents a new gene therapy-based strategy for the treatment of inherited diseases, such as Fanconi anemia (FA). Because FA can be generated by multiple mutations in up to 19 different genes, we focused our interests in the insertion of therapeutic FA genes in safe harbor loci, since this approach could serve as a platform for treating all FA subtypes and pathogenic mutations. In previous studies we demonstrated the feasibility of correcting the phenotype of hematopoietic progenitors through the genome editing and reprogramming of fibroblasts from FA patients. Because of the limited repopulating properties so far reported for iPSC-derived hematopoietic precursor cells, here we aimed to investigate the possibility of conducting a similar gene targeting approach using human primary CD34+ cells. In a first set of experiments we investigated the efficiency of targeting the AAVS1 locus in cord blood HSPCs. Pre-stimulated CD34+ cells were transduced with integrase-defective lentiviral vectors (IDLV) encoding an EGFP reporter gene under the control of the PGK promoter, flanked by sequences homologous to the AAVS1 target site. The cells were then nucleofected with AAVS1 -specific ZFN mRNAs. The average targeting efficiencies ranged from 10-20% both in in vitro cultured cells and NSG mice-repopulating cells. In subsequent experiments, the EGFP reporter gene was replaced by the hFANCA therapeutic gene. Gene targeting experiments conducted in lymphoblast cell lines and primary CD34+ cells from FA-A patients reverted the characteristic hypersensitivity of FA cells to mitomycin C (MMC), and also restored the formation of FANCD2 foci after DNA damage, evidencing the phenotypic correction of these cells. Our data confirm the efficacy of AAVS1-gene targeting in hHSPCs and demonstrate the feasibility of conducting these approaches in primary CD34+ cells from FA patients.

Regulation of transcription factor IRF8 in myeloid progenitors is a critical checkpoint for formation of defective myeloid cells in cancer.

Abstract Defectivemyeloid cells are commonly observed across a large range of solid cancers. These defects contribute to immune suppression and tumor progression tometastatic disease. Early studies focused on characterizing such myeloid-derivedsuppressor cells (MDSC) in peripheral tissues and the tumor microenvironment, but molecular processes underlying their development remained unclear. Bone marrowis the major site for normal myeloid production and is known to respond to signalsfrom the periphery. Thus, we hypothesizedthat tumors perturb bone marrow myelopoiesis, resulting in production of MDSC. During myelopoiesis, transcription factor IRF8 expression within granulocyte/monocyteprogenitors (GMP) determines differentiation such that high IRF8 favorsmonocytes while low IRF8 favors granulocytes. We previously revealed that IRF8 suppressionby tumor-derived factors leads to accumulation of MDSC, but exactly when andwhere IRF8 is compromised was unknown. Here we utilize a novel mouse model expressingan IRF8-EGFP fusion protein to investigate specific changes in IRF8 during myelopoiesisin both orthotopic and spontaneous mammary tumor progression. Our results show that:1) GMP can be divided into IRF8hi and IRF8lo expressingpopulations corresponding to monocytic and granulocytic progenitor phenotypes,2) IRF8lo GMP preferentially expand with increasing tumor size andincreasing G-CSF serum concentration, and 3) enforced IRF8 expression restrainsthis expansion and reduces the frequency of myeloid suppressors in theperiphery. Altogether, these data identify modulation of IRF8 in myeloidprogenitors as a consequence of tumor development and potential target fortherapeutic intervention.

Embryonic stem cell-derived B-1 cells engraft in immunodeficient mice, recapitulating YS B lymphopoiesis.

Abstract It has been reported that B cells are produced from embryonic stem cells (ESCs) when co-cultured with OP9 stromal cells. However, these ESC-derived B cells have not been well characterized; it is unknown whether they belong to the B-1 or B-2 lineage and whether they are transplantable into immunodeficient mice. We previously reported that the first wave of B lymphopoiesis occurs in the extra-embryonic yolk sac (YS) at the time prior to hematopoietic stem cell (HSC) emergence and these cells belong to the B-1 cell lineage. YS-derived B progenitor cells display a AA4.1+CD19+B220dim B-1 specific progenitor phenotype, are transplantable into immunodeficient mice, and secrete natural antibodies; all key features of B-1 cells. Since differentiation of ESC into blood cells recapitulates YS hematopoiesis, we hypothesized that B progenitor cells produced from mouse ESCs in vitro belong to the B-1 cell lineage. We co-cultured ESCs with OP9 stromal cells and generated Flk1+ cells. Isolated Flk1+ cells were replated onto OP9 stromal cells, and after 9–12 days of co-culture, AA4.1+CD19+B220+ cells were detected. We transplanted these cells into sublethally irradiated immunodeficient neonates. Donor-derived B-1 cells were detected in the recipient peritoneal cavity up to 6 months after transplantation. Furthermore, ESC-derived B-1 cells produced anti-phosphorylcholine antibodies detected by Elispot assay, showing the ability of these innate B-1 cells to produce natural antibodies. Thus, we have demonstrated that ESC-derived B cells engraft in vivo as functional B -1 cells secreting natural antibodies. These B cell developmental processes recapitulate B lymphopoiesis in the YS and might be utilized for innate immune cell therapy in the future.