Lineage-committed Progenitor Cells Research Articles

20 Large Burn Injury Leads to Pathological Alteration of Hematopoietic Stem and Progenitor Cell Lineage Commitment

20 Large Burn Injury Leads to Pathological Alteration of Hematopoietic Stem and Progenitor Cell Lineage Commitment

Amphiregulin Switches Progenitor Cell Fate for Lineage Commitment During Gastric Mucosal Regeneration

Background & AimsIsthmic progenitors, tissue-specific stem cells in the stomach corpus, maintain mucosal homeostasis by balancing between proliferation and differentiation to gastric epithelial lineages. The progenitor cells rapidly adopt an active state in response to mucosal injury. However, it remains unclear how the isthmic progenitor cell niche is controlled during the regeneration of damaged epithelium. MethodsWe recapitulated tissue recovery process after acute mucosal injury in the mouse stomach. BrdU incorporation was utilized to trace newly generated cells during the injury and recovery phases. To define epithelial lineage commitment process during recovery, we performed single cell RNA-sequencing (scRNA-seq) on epithelial cells from the mouse stomachs. We validated the effects of amphiregulin (AREG) on mucosal recovery, utilizing recombinant AREG treatment or AREG deficient mice. ResultsWe determined that an EGFR ligand, AREG, can control progenitor cell lineage commitment. Based on the identification of lineage-committed subpopulations in the corpus epithelium through scRNA-seq and BrdU incorporation, we showed that isthmic progenitors mainly transition into short-lived surface cell lineages, but are less frequently committed to long-lived parietal cell lineages in homeostasis. However, mucosal regeneration after damage directs the lineage commitment of isthmic progenitors towards parietal cell lineages. During recovery, AREG treatment promoted repopulation with parietal cells, while suppressing surface cell commitment of progenitors. In contrast, TGF-alpha did not alter parietal cell regeneration, but did induce expansion of surface cell populations. AREG deficiency impairs parietal cell regeneration, but increases surface cell commitment. ConclusionsThese data demonstrate that different EGF receptor ligands can distinctly regulate isthmic progenitor-driven mucosal regeneration and lineage commitment.

A Mouse Model of Zhu-Tokita-Takenouchi-Kim Syndrome Reveals Indispensable Son Functions in Myeloid-Lymphoid Lineage Balance and Terminal Differentiation

SON is a DNA- and RNA-binding protein mainly localized in nuclear speckles. Compromised SON function leads to aberrant and alternative RNA splicing, particularly for transcripts bearing weak splice sites. SON also binds to DNA and suppresses H3K4me3 modifications at transcription start sites by inhibiting the MLL1/2 methyltransferase complex. More recent studies suggest that SON serves as the core of nuclear speckles, and SON is required for the association between nuclear speckles and p53 target genes to boost RNA expression. Therefore, SON governs the expression of a myriad of target genes by regulating transcription and RNA splicing as well as nuclear speckle assembly, yet exerts its effects on specific sets of genes. However, the role of SON in organ development and hematopoiesis remains poorly understood. Recently, we identified that heterozygous loss-of-function (LoF) mutations in the SON gene cause a neurodevelopmental disorder with multi-organ anomalies in human patients. This syndrome has been designated as a novel rare disease named Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome. The ZTTK SON-Shine Foundation, a family-initiated non-profit organization, was launched, and through this foundation, we work with families to characterize clinical symptoms associated with SON LoF. Among the clinical symptoms observed in individuals with ZTTK syndrome, a noticeable feature is a wide range of hematological abnormalities, including bone marrow failure, high mean corpuscular volume (MCV), polycythemia, severe anemia, and thrombosis. Furthermore, immunoglobulin deficiency, poor responses to vaccines, and frequent infections were reported, implicating SON LoF in various hematopoietic abnormalities. To study how heterozygous LoF of SON affects organ development and causes clinical features associated with ZTTK syndrome, we created a mouse model of Son haploinsufficiency ( Son +/-). While complete knockout of Son ( Son -/-) led to embryonic lethality, Son +/- mice were viable and recapitulated many clinical features of human ZTTK syndrome patients. The Son +/- mice showed severe growth retardation, signs of cognitive impairment, skeletal abnormalities, kidney agenesis/hypoplasia, and, importantly, hematopoietic abnormalities, such as high MCV, low lymphocyte counts, and immunoglobulin deficiency. To examine the hematopoietic features found in ZTTK syndrome patients are due to SON LoF, we further examined hematopoiesis of our Son +/- mice. We found that Son +/- fetal liver as well as adult mouse bone marrow contain a reduced size of the early-stage hematopoietic stem and progenitor cell (HSPC) pool (marked as Lin-Sca1+cKit+: LSK). Analysis of LSK subpopulations revealed that Son +/- mice have myeloid-lymphoid lineage imbalance at the stage of multipotent progenitor (MPP), with increased megakaryocyte/erythroid lineage-biased MPPs (MPP2) while lymphoid lineage-biased MPPs (MPP4) are decreased. In addition, erythroid terminal differentiation and B cell maturation were impaired in Son +/- mice. Single-cell RNA-sequencing of mouse bone marrow Lin-cKit+ cells and transcriptome-based clustering also revealed that Son haploinsufficiency leads to a reduction in lymphoid lineage-committed progenitor cells and decreased expression of lymphoid lineage/B cell development genes, while erythroid lineage-promoting genes were aberrantly activated in myeloid lineage progenitors. Deep bulk RNA-sequencing and alternative splicing analysis identified that that Son haploinsufficiency caused alternative splicing of chromatin modifiers genes and epigenetic regulator genes. Furthermore, from our ATAC-sequencing analysis, we found that Son haploinsufficiency causes decreased chromatin accessibility in the gene loci critical for lymphoid lineage specification. Taken together, our Son +/- mouse model revealed that the normal level of Son is required to achieve the sufficient expression levels of the genes directing lymphoid lineage specification, B cell lineage development, and erythroid terminal differentiation. Our study demonstrates the indispensable roles of SON in multiple stages of hematopoiesis and provides valuable insight into the clinical significance of abnormal SON levels in hematopoietic diseases.

Physioxia improves the selectivity of hematopoietic stem cell expansion cultures.

Hematopoietic stem cells (HSCs) are a rare hematopoietic cell type that can entirely reconstitute the blood and immune systems following transplantation. Allogeneic HSC transplantation (HSCT) is used clinically as a curative therapy for a range of hematolymphoid diseases, but remains a high-risk therapy due to potential side effects including poor graft function and graft-vs-host disease (GvHD). Ex vivo HSC expansion has been suggested as an approach to improve hematopoietic reconstitution from low-cell dose grafts. Here, we demonstrate that we can improve the selectivity of polyvinyl alcohol (PVA)-based mouse HSC cultures through the use of physioxic culture conditions. Single-cell transcriptomic analysis confirmed inhibition of lineage-committed progenitor cells in physioxic cultures. Long-term physioxic expansion also afforded culture-based ex vivo HSC selection from whole bone marrow, spleen, and embryonic tissues. Furthermore, we provide evidence that HSC-selective ex vivo cultures deplete GvHD-causing T cells and that this approach can be combined with genotoxic-free antibody-based conditioning HSCT approaches. Our results offer a simple approach to improve PVA-based HSC cultures and the underlying molecular phenotype, as well as highlight the potential translational implications of selective HSC expansion systems for allogeneic HSCT.

Mesenchymal Stem Cells Derived from Umbilical Cord Blood Having Excellent Stemness Properties with Therapeutic Benefits - a New Era in Cancer Treatment.

Mesenchymal stem cells (MSCs) are the most promising candidates for cellular therapies, and most therapeutic applications have focused on MSCs produced from adult bone marrow, despite mounting evidence that MSCs are present in a wide range of conditions. Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells, but its therapeutic potential extends beyond the hematopoietic component, which also suggests solid organ regenerative potential. With potential ranging from embryonic- like to lineage-committed progenitor cells, many different stems and progenitor cell populations have been postulated. MSC is currently inferred by numerous clinical applications for human UCB. As stem cell therapy kicks off some new research and these cells show such a boon to stem cell therapy, it is nevertheless characteristic that the prospect of UCB conservation is gaining momentum. Taken together, the experience described here shows that MSCs derived from UCB are seen as attractive therapeutic candidates for various human disorders including cancer. It is argued that a therapeutic stem cell transplant, using stem cells from UCB, provides a reliable repository of early precursor cells that can be useful in a large number of different conditions, considering issues of safety, availability, transplant methodology, rejection, and side effects. In particular, we focus on the concept of isolation and expansion, comparing the phenotype with MSC derived from the UCB, describing the ability to differentiate, and lastly, the therapeutic potential concerning stromal support, stemness characteristic, immune modulation, and cancer stem cell therapy. Thus it is an overview of the therapeutic application of UCB derived MSCs, with a special emphasis on cancer. Besides, the current evidence on the double-edged sword of MSCs in cancer treatment and the latest advances in UCB-derived MSC in cancer research will be discussed.

Progenitor cells derived from gene-engineered human induced pluripotent stem cells as synthetic cancer cell alternatives for in vitro pharmacology.

Limitations in genetic stability and recapitulating accurate physiological disease properties challenge the utility of patient-derived (PD) cancer models for reproducible and translational research. A portfolio of isogenic human induced pluripotent stem cells (hiPSCs) with different pan-cancer relevant oncoprotein signatures followed by differentiation into lineage-committed progenitor cells was genetically engineered. Characterization on molecular and biological level validated successful stable genetic alterations in pluripotency state as well as upon differentiation to prove the functionality of our approach. Meanwhile proposing core molecular networks possibly involved in early dysregulation of stem cell homeostasis, the application of our cell systems in comparative substance testing indicates the potential for cancer research such as identification of augmented therapy resistance of stem cells in response to activation of distinct oncogenic signatures.

Single Cell Analysis Elucidates the Maturation of Human Stem and Progenitor Cell Function from Fetal through Adult Hematopoiesis

Hematopoiesis continually replenishes the supply of circulating blood cells from embryonic development through the entirety of human lifespan. Although all hematopoietic lineages are produced throughout life, biases in lineage output occur at various stages, including lymphoid bias in childhood and myeloid bias in adulthood. Furthermore, many blood disorders demonstrate marked biases in age of onset, such as bone marrow failure disorders, clonal hematopoiesis of indeterminate potential, and numerous hematologic malignancies. A lack of insight into the normal physiologic changes occurring in the hematopoietic stem and progenitor cell (HSPC) compartments during development and maturation fundamentally limits our understanding of how age biased blood disorders arise. Two major unresolved questions are: (1) what changes in the molecular regulation of hematopoietic lineage commitment occur over the course of human life? And (2) are certain HSPC states present only during specific ages of life, and if so, do age-specific HSPCs have distinct biology?To address these questions we performed single cell RNA sequencing (scRNAseq) on human HSPCs from first and second trimester fetal liver hematopoiesis, and bone marrow hematopoiesis spanning childhood into mature adulthood. In total, HSPC samples were obtained from 14 distinct human donors. Dimensionality reduction and marker gene analysis identified uncommitted hematopoietic stem cells (HSCs) and the developmental trajectories of each lineage emanating from multipotent HSCs. We then identified the genes activated upon commitment to each hematopoietic lineage during fetal, childhood, and mature adult hematopoiesis using the Population Balance Analysis and Stationary Optimal Transport algorithms, followed by Elastic Net gene regression. For each lineage we determined the putative transcription factor network that is consistently active in driving commitment to that lineage throughout life, but surprisingly also found the existence of adjunctive transcription factor networks that only drove lineage commitment at specific ages.We next used unbiased clustering of scRNAseq data to identify 21 distinct subtypes in the HSPC compartment across human life. Using marker gene analysis and singleCellNet algorithm comparisons to an existing human adult bone marrow scRNAseq data set, we hierarchically ordered and annotated these HSPC subtypes ranging from uncommitted HSCs to lineage committed progenitor cells. We found that cellular distribution within the HSPC compartment amongst these subtypes varied markedly throughout human lifetime, with higher representation of HSCs in fetal life, predominance of lymphoid progenitors in childhood, and higher representation of myeloid progenitors in adulthood. Focusing on the distribution of cells among HSC subtypes over human life, we identified an HSC subtype exclusive to mid-gestation that was not present in early fetal or postnatal timepoints. This HSC subtype had a characteristic immunophenotype and was enriched for expression of early response transcription factors and mRNA decay factors. We functionally validated that this mid-gestation-specific HSC subtype was phenotypically unique using colony formation assays and xenotransplantation. Mid-gestation-specific HSCs were more clonogenic with a greater number of multi-lineage outcomes, and also demonstrated increased multilineage engraftment capacity compared to other HSC subtypesOur findings reveal that the intrinsic biology of hematopoietic lineage commitment fundamentally changes over the course of the human lifetime, and define and validate age-specific HSPC subtypes. In particular, the biology of the mid-gestation-specific HSC we identified has potential applications for improving engraftment and multi-lineage reconstitution in hematopoietic cell transplantation. DisclosuresRegev: Genentech: Current Employment; Celsius Therapeutics: Current equity holder in publicly-traded company, Other: Co-founder; Immunitas: Current equity holder in publicly-traded company; ThermoFisher Scientific: Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Neogene Therapeutics: Membership on an entity's Board of Directors or advisory committees; Asimov: Membership on an entity's Board of Directors or advisory committees.

NAD+ augmentation with nicotinamide riboside improves lymphoid potential of Atm\u2212/\u2212 and old mice HSCs

NAD+ supplementation has significant benefits in compromised settings, acting largely through improved mitochondrial function and DNA repair. Elevating NAD+ to physiological levels has been shown to improve the function of some adult stem cells, with implications that these changes will lead to sustained improvement of the tissue or system. Here, we examined the effect of elevating NAD+ levels in models with reduced hematopoietic stem cell (HSC) potential, ATM-deficient and aged WT mice, and showed that supplementation of nicotinamide riboside (NR), a NAD+ precursor, improved lymphoid lineage potential during supplementation. In aged mice, this improved lymphoid potential was maintained in competitive transplants and was associated with transcriptional repression of myeloid gene signatures in stem and lineage-committed progenitor cells after NR treatment. However, the altered transcriptional priming of the stem cells toward lymphoid lineages was not sustained in the aged mice after NR removal. These data characterize significant alterations to the lineage potential of functionally compromised HSCs after short-term exposure to NR treatment.

The Inhibitor of MyoD Family A Promotes Terminal Myeloid Differentiation

Abstract The differentiation of hematopoietic stem and progenitor cells is tightly regulated to maintain immune and blood homeostasis while also responding to injury and immunological insult. Myeloid lineage-committed progenitor cells are highly proliferative, but do not self-renew and must be continually replenished by the differentiation of hematopoietic stem cells. The process of terminal differentiation is controlled by various cell-intrinsic and - extrinsic factors, and the interactions between these factors remain not well understood. We hypothesize that the Inhibitor of MyoD Family A (I-mfa) protein is a novel regulator of myeloid lineage differentiation through its interaction with other cell-intrinsic factors. To examine this, mice which lack I-mfa expression (I-mfanull) were compared to wild type (WT) 129Sv age- and sex-matched controls. I-mfanull animals had reduced spleen size (both in weight and total cellularity), but had preserved relative proportions of B, T, and myeloid cells as analyzed by flow cytometry when compared to WT. Analysis of the bone marrow (BM) myeloid lineage-committed progenitors showed an accumulation of common myeloid progenitors (CMP) and granulocyte-macrophage progenitors (GMP) in I-mfanull animals when compared to WT. In serial colony forming unit assays I-mfanull BM produced fewer mature colonies and continued to produce colonies after 5 passages, suggesting aberrant self-renewal. Using myeloid cell lines lacking or overexpressing I-mfa, the molecular mechanism by which I-mfa acts is being elucidated. These results taken together suggest I-mfa acts to promote differentiation of myeloid cells, an effect which may be important in the treatment of myeloid neoplasms or bone marrow failure.

Evolutionary selection of alleles in the melanophilin gene that impacts on prostate organ function and cancer risk.

Background and objectivesSeveral hundred inherited genetic variants or SNPs that alter the risk of cancer have been identified through genome-wide association studies. In populations of European ancestry, these variants are mostly present at relatively high frequencies. To gain insight into evolutionary origins, we screened a series of genes and SNPs linked to breast or prostate cancer for signatures of historical positive selection.MethodologyWe took advantage of the availability of the 1000 genome data and we performed genomic scans for positive selection in five different Caucasian populations as well as one African reference population. We then used prostate organoid cultures to provide a possible functional explanation for the interplay between the action of evolutionary forces and the disease risk association.ResultsVariants in only one gene showed genomic signatures of positive, evolutionary selection within Caucasian populations melanophilin (MLPH). Functional depletion of MLPH in prostate organoids, by CRISPR/Cas9 mutation, impacted lineage commitment of progenitor cells promoting luminal versus basal cell differentiation and on resistance to androgen deprivation.Conclusions and implicationsThe MLPH variants influencing prostate cancer risk may have been historically selected for their adaptive benefit on skin pigmentation but MLPH is highly expressed in the prostate and the derivative, positively selected, alleles decrease the risk of prostate cancer. Our study suggests a potential functional mechanism via which MLPH and its genetic variants could influence risk of prostate cancer, as a serendipitous consequence of prior evolutionary benefits to another tissue.Lay SummaryWe screened a limited series of genomic variants associated with breast and prostate cancer risk for signatures of historical positive selection. Variants within the melanophilin (MLPH) gene fell into this category. Depletion of MLPH in prostate organoid cultures, suggested a potential functional mechanism for impacting on cancer risk, as a serendipitous consequence of prior evolutionary benefits to another tissue.

Purification of Human CD34+CD90+ HSCs Reduces Target Cell Population and Improves Lentiviral Transduction for Gene Therapy

Hematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant, and infectious diseases. We have shown in a nonhuman primate gene therapy and transplantation model that the CD34+CD90+ cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. In this study, we show the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy. Alternative HSC enrichment strategies include the purification of CD133+ cells or CD38low/– subsets of CD34+ cells from human blood products. We directly compared these strategies to the isolation of CD90+ cells using a good manufacturing practice (GMP) grade flow-sorting protocol with clinical applicability. We show that CD90+ cell selection results in about 30-fold fewer target cells in comparison to CD133+ or CD38low/– CD34+ hematopoietic stem and progenitor cell (HSPC) subsets without compromising the engraftment potential in vivo. Single-cell RNA sequencing confirmed nearly complete depletion of lineage-committed progenitor cells in CD90+ fractions compared to alternative selections. Importantly, lentiviral transduction efficiency in purified CD90+ cells resulted in up to 3-fold higher levels of engrafted gene-modified blood cells. These studies should have important implications for the manufacturing of patient-specific HSC gene therapy and gene-engineered cell products.

Slug and E-Cadherin: Stealth Accomplices?

During physiological epithelial-mesenchymal transition (EMT), which is important for embryogenesis and wound healing, epithelial cells activate a program to remodel their structure and achieve a mesenchymal fate. In cancer cells, EMT confers increased invasiveness and tumor-initiating capacity, which contribute to metastasis and resistance to therapeutics. However, cellular plasticity that navigates between epithelial and mesenchymal states and maintenance of a hybrid or partial E/M phenotype appears to be even more important for cancer progression. Besides other core EMT transcription factors, the well-characterized Snail-family proteins Snail (SNAI1) and Slug (SNAI2) play important roles in both physiological and pathological EMT. Often mentioned in unison, they do, however, differ in their functions in many scenarios. Indeed, Slug expression does not always correlate with complete EMT or loss of E-cadherin (CDH1). For example, Slug plays important roles in mammary epithelial cell progenitor cell lineage commitment and differentiation, DNA damage responses, hematopoietic stem cell self-renewal, and in pathologies such as pulmonary fibrosis and atherosclerosis. In this Perspective, we highlight Slug functions in mammary epithelial cells and breast cancer as a “non-EMT factor” in basal epithelial cells and stem cells with focus reports that demonstrate co-expression of Slug and E-cadherin. We speculate that Slug and E-cadherin may cooperate in normal mammary gland and breast cancer/stem cells and advocate for functional assessment of such Slug+/E-cadherinlow/+ (SNAI2+/CDH1low/+) “basal-like epithelial” cells. Thus, Slug may be regarded as less of an EMT factor than driver of the basal epithelial cell phenotype.

Paradigms that define lung epithelial progenitor cell fate in development and regeneration.

Throughout the lifespan, lung injury impedes the primary critical function essential for life-respiration. To repair quickly and efficiently is critical and is orchestrated by a diverse repertoire of progenitor cells and their niche. This review incorporates knowledge gained from early studies in lung epithelial morphogenesis and cell fate and explores its relevance to more recent findings of lung progenitor and stem cells in development and regeneration. Cell fate in the lung is organized into an early specification phase and progressive differentiation phase in lung development. The advent of single cell analysis combined with lineage analysis and projections is uncovering new functional cell types in the lung providing a topographical atlas for progenitor cell lineage commitment during development, homeostasis, and regeneration. Lineage commitment of lung progenitor cells is spatiotemporally regulated during development. Single cell sequencing technologies have significantly advanced our understanding of the similarities and differences between developmental and regenerative cell fate trajectories. Subsequent unraveling of the molecular mechanisms underlying these cell fate decisions will be essential to manipulating progenitor cells for regeneration.

Azacytidine Inhibits Megakaryopoiesis Via the Induction of Immunogenic RNA Species and Activation of Type-I Interferon Signaling

Management of hematologic disease- and therapy-related thrombocytopenia remains a serious clinical issue, especially in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The ribonucleoside and DNA-demethylating agent azacytidine (AZA), has proven useful for the treatment of patients with MDS and AML not eligible for stem cell transplantation. While low-dose AZA therapy induces clinical remissions in up to 50% of treated patients, it comes at the cost of aggravating pre-existing thrombocytopenia which is observed in a subset of patients; this can lead to increased bleeding and bleeding-associated mortality, and importantly, often requires dose modifications and delays of therapy. Thus, identification of strategies alleviating ineffective megakaryopoiesis will likely lead to increased therapeutic efficacy for patients with MDS/AML. Eltrombopag (EP), a second-generation small molecule thrombopoietin receptor (TPO-R) agonist was effective in raising platelet counts in patients with MDS as a single agent, as well as in combination with certain standard of care therapies. However, it failed to stimulate platelet production during the first four cycles of AZA treatment as uncovered by a recent phase III placebo-controlled clinical study (SUPPORT; NCT02158936). The goals of this study were to identify the cellular and molecular underpinnings of AZA-associated inhibition of megakaryopoiesis and to assess the ineffectiveness of EP in mitigating AZA treatment-associated thrombocytopenia. Our results demonstrate that at a clinically-equivalent and non-cytotoxic dose, AZA rapidly induces transient activation of interferon type I (IFN-I) signaling in various hematopoietic cell types, including stem and lineage-committed progenitor cells (HSPCs). We detected IFNα and IFNβ production and release using ELISA and intracellular flow cytometry on primary total mononuclear cell- and purified CD34-positive HSPC populations derived from cord blood, bone marrow from healthy volunteers or patients with MDS/AML. AZA-mediated activation of Type I IFNs in healthy control- and MDS/AML cells was preceded by an accumulation of double-stranded RNA (dsRNA) species and decreased total RNA cytosine methylation measured by immunocytochemistry and intracellular FACS analysis; this suggested that AZA triggered the accumulation of immunogenic RNA species which elicit an IFN-I response. In support, we found Toll like receptor 3 (TLR3) activation and phosphorylation of STAT1 in CD34+ HPSC, along with premature activation of Suppressor of Cytokine Signaling 1 (SOCS1), a well-known JAK/STAT-dependent signaling attenuator. This rapid AZA-induced viral mimicry response led to abrogation of thrombopoietin (TPO) or EP-stimulated TPO-R signaling and inhibition of ex vivo megakaryocyte progenitor proliferation quantified by colony formation in semi-solid medium. Importantly, inhibition of IFN-I signal activation using the JAK3 inhibitor decernotinib, the IFNα/β-blocking peptide, B18R, or RNA interference-mediated knock-down of SOCS1 counteracted the inhibitory effects of AZA on TPO-R stimulation and restored megakaryopoiesis. Given these observations, we pre-clinically tested a revised treatment protocol, in which primary cells were first exposed to AZA for four days followed by TPO-R stimulation using TPO or EP. This new treatment strategy alleviated AZA's inhibitory effects at the molecular and cellular levels, demonstrating that upon resolution of the AZA-mediated vial mimicry response, EP and TPO can effectively stimulate TPO-R signaling and megakaryopoiesis. Together, our data reveal a mechanistic basis of AZA-mediated inhibition of megakaryopoiesis in patients with MDS/AML. Additionally, we show that EP cannot overcome the megakaryopoiesis-inhibitory effects of acute IFN-I signaling activation upon AZA exposure. Findings of our study are consistent with and provide a molecular explanation for the observations made in the context of the SUPPORT study. In the future, it will be critical to better understand and potentially counteract the megakaryopoiesis-inhibitory effects by IFN-I pathway activation upon AZA therapy in patients with MDS/AML. Disclosures Okoye-Okafor: Novartis Pharmaceuticals: Research Funding. Pallaud:Novartis Pharmaceuticals: Employment. Marques Ramos:Novartis Pharmaceuticals: Employment. Verma:Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding. Will:Novartis Pharmaceuticals: Research Funding.

ITERATIVE AND PLEIOTROPIC ROLES OF ENDOTHELIAL CELLS IN HUMAN HEMATOPOIETIC ERYTHROPOIESIS

Local tissue microenvironments regulate directly the hematopoietic development and cell fate specification of both stem cells and lineage-committed progenitor cells. The identification of hematopoietic niches and the mechanisms by which they regulate cell fate specification offers the opportunity to better understand tissue homeostasis and regeneration. Hematopoietic stem cells (HSCs) and lineage-committed progenitor cells are closely associated with blood vessels during hematopoietic development. Recent studies have confirmed the essential role of endothelial cells (ECs) in the maintenance and lineage-specification of HSCs. However, the precise mechanisms through which ECs regulate hematopoietic development are not well understood. Particularly, we know little about which stage in the development ECs regulate hematopoietic progenitor cells. Here we identified that ECs promote erythropoiesis in a pleiotropic and iterative manner. We cultured CD34+ human hematopoietic stem and progenitor cells (HSPCs) in the presence of vascular ECs. Following in vitro differentiation, we measured the number of various lineage cells using surface marker detection. Among hematopoietic cells, we found that both CD34+CD38- HSPCs and CD71+CD235A+ erythroblasts were increased. Transwell culture system validated that ECs facilitate erythropoiesis via soluble factors. Noteworthy, primitive ECs derived from pluripotent stem cells increased the number of HSPCs, while mature ECs derived from umbilical cord increased the number of erythroblasts. The results demonstrate that primitive and mature ECs facilitate erythropoiesis at both the HSPC and erythroblast level, respectively, and suggest that the chronicity of hematopoiesis matches the niche. Our study proposes iterative and pleiotropic roles of ECs in human hematopoietic erythropoiesis.

The Best for the Most Important: Maintaining a Pristine Proteome in Stem and Progenitor Cells.

Pluripotent stem cells give rise to reproductively enabled offsprings by generating progressively lineage-restricted multipotent stem cells that would differentiate into lineage-committed stem and progenitor cells. These lineage-committed stem and progenitor cells give rise to all adult tissues and organs. Adult stem and progenitor cells are generated as part of the developmental program and play critical roles in tissue and organ maintenance and/or regeneration. The ability of pluripotent stem cells to self-renew, maintain pluripotency, and differentiate into a multicellular organism is highly dependent on sensing and integrating extracellular and extraorganismal cues. Proteins perform and integrate almost all cellular functions including signal transduction, regulation of gene expression, metabolism, and cell division and death. Therefore, maintenance of an appropriate mix of correctly folded proteins, a pristine proteome, is essential for proper stem cell function. The stem cells' proteome must be pristine because unfolded, misfolded, or otherwise damaged proteins would interfere with unlimited self-renewal, maintenance of pluripotency, differentiation into downstream lineages, and consequently with the development of properly functioning tissue and organs. Understanding how various stem cells generate and maintain a pristine proteome is therefore essential for exploiting their potential in regenerative medicine and possibly for the discovery of novel approaches for maintaining, propagating, and differentiating pluripotent, multipotent, and adult stem cells as well as induced pluripotent stem cells. In this review, we will summarize cellular networks used by various stem cells for generation and maintenance of a pristine proteome. We will also explore the coordination of these networks with one another and their integration with the gene regulatory and signaling networks.

Discovery and Development of MEDI7247, a Novel Pyrrolobenzodiazepine (PBD)-Based Antibody Drug Conjugate Targeting ASCT2, for Treating Hematological Cancers

The neutral amino acid transporter, ASCT2, is frequently overexpressed in several cancers to sustain “glutamine addiction” of cancer cells. High expression of ASCT2 is often associated with poor disease prognosis. Immuno-histochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) tissue samples revealed high prevalence of membranous ASCT2 expression in several hematological cancers, including MM, AML, and DLBCL summarized in Table1. ASCT2 expression was predominantly on the plasma membrane of the carcinoma cells. ASCT2 staining was observed in almost all the samples with high positivity (>2+ and in >50% of tumor cells) in 98, 74 and 55% of MM, AML and DLBCL samples respectively. Additionally, flow cytometry analyses suggest significantly high expression of ASCT2 in bone marrow (BM) samples from AML and MM patients in comparison to BM from healthy donors. Also, our data suggest relatively less expression of ASCT2 in LT-HSC (long term hematopoietic stem cells) in comparison to prominent myeloid-associated marker CD33. In contrast, we found similar expression profile of ASCT2 and CD33 in downstream lineage-committed progenitor cells, such as MPP (multi potent progenitors) and CP (common progenitors). Furthermore, IHC evaluated across normal tissues suggest restricted ASCT2 expression in the normal tissues of vital organs.Broad expression across various unmet cancers and restricted expression in normal tissues warrant ASCT2 as an attractive candidate for an antibody drug conjugate. MEDI7247 is a novel investigational antibody-drug conjugate (ADC) comprising an anti-ASCT2 human monoclonal antibody site-specifically conjugated to pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker, with a drug to antibody ratio (DAR) of 2. MEDI7247 specifically binds to the cell surface ASCT2 while exhibiting no affinity to the other members of the family, including ASCT1. Following binding to the extracellular region of ASCT2 antigen, MEDI7247 is internalized and trafficked to the lysosomes to subsequently release the PBD warhead. The release of PBD induces DNA damage and results in tumor cell death.MEDI7247 shows potent in vitro cytotoxic killing in several heme cancer cell lines (IC50 range of 0.05 ng/ml to ~65 ng/ml) with variable levels of ASCT2 expression (e.g. H929 (high) ~ 1.1 X106 copies/cell; Nomo (low) ~ 1.3 X105 copies/cell). In vivo anti-tumor activity of MEDI7247 was evaluated in a panel of hematological tumor models (CDx) representing AML, MM, DLBCL, cALL, and Burkitt's lymphoma. Briefly, MEDI7247 (dosed: single dose or Q1Wx4) demonstrated a significant survival advantage in disseminated or subcutaneous tumor models, when compared to the untreated control or standard of care (SOC) at the various dose levels examined: 0.05, 0.1 and 0.4 mg/kg, respectively. Likewise, MEDI7247 was also tested in disseminated AML PDX (ASCT2-low) models. Significant improvement in survival was observed at both 0.1 and 0.4 mg/kg with the higher dose level extending survival by >80 days and PDX models showed robust antitumor effect and significant survival benefit compared to standard of care (SOC).We used flow cytometry sorting of CD34+ and CD38+ populations from AML BM and ran colony forming assays in methocult culture conditions to confirm that CD34+CD38+ cells are leukemic stem cells (LSC) of AML BM samples. Furthermore, we confirmed high expression of ASCT2 in LSC and in the bulk population of AML BM. Similar analyses suggest relatively high expression of ASCT2 in MM plasma cells (CD138+CD19-) and modest to low expression in MM stem cells (CD19+ CD138-). This may yield opportunity for more durable clinical response in AML, genetically heterogeneous diseases. Furthermore, MEDI7247 demonstrated acceptable safety profile in toxicity studies with non-human primates to support first in human trials.In conclusion, based on its combined efficacy and safety, MEDI7247, a first-in class ADC is currently being evaluated in clinic for the treatment of ASCT2 positive hematological malignancies (NCT03106428). [Display omitted] DisclosuresPore:Medimmune: Employment. Schifferli:Medimmune: Employment. Monks:Medimmune: Employment. Tammali:Medimmune: Employment. Borrok:Medimmune: Employment. Hurt:Medimmune: Employment. Flynn:Medimmune: Employment. Rebelatto:Medimmune: Employment. Townsley:Medimmue: Employment. Hinrichs:Medimmune: Employment. Dixit:Medimmune: Employment. Coats:Medimmune: Employment. Herbst:Medimmune: Employment. Tice:Medimmune: Employment.

Biomaterials as cell carriers for augmentation of adipose tissue-derived stromal cell transplantation.

Adipose tissue-derived stromal cells (ADSCs) contain lineage-committed progenitor cells that have the ability to differentiate into various cell types that may be useful for autologous cell transplantation to correct defects of skin, adipose, cartilage, bone, tendon, and blood vessels. The multipotent characteristics of ADSCs, as well as their abundance in the human body, make them an attractive potential resource for wound repair and applications to tissue engineering. ADSC transplantation has been used in combination with biomaterials, including cell sheets, hydrogel, and three-dimensional (3D) scaffolds based on chitosan, fibrin, atelocollagen, and decellularized porcine dermis, etc. Furthermore, low molecular weight heparin/protamine nanoparticles (LH/P NPs) have been used as an inducer of ADSC aggregation. The tissue engineering potential of these biomaterials as cell carriers is increased by the synergistic relationship between ADSCs and the biomaterials, resulting in the release of angiogenic cytokines and growth factors. In this review article, we describe the advantages of ADSC transplantation for tissue engineering, focusing on biomaterials as cell carriers which we have studied.

3016 - JARID2 Restricts Self-Renewal Capacity in Hematopoietic Progenitors and is A Myeloid Tumor Suppressor

The most common genetic mutations in myeloproliferative neoplasms (MPN) occur in JAK2, MPL and CALR, while additional mutations in genes such as TET2, IDH1/2, and NRAS drive transformation to secondary acute myeloid leukemia (sAML). Genome sequencing studies identified chromosomal deletions of JARID2, a Polycomb Repressive Complex 2 (PRC2) co-factor, in post-MPN sAML. To investigate Jarid2 as a tumor suppressor, we utilized an inducible mouse model of Jak2V617F combined with floxed alleles of Jarid2 and the inducible Mx1-CRE. The absence of Jarid2 accelerated MPN, with a median survival of 23 and 43 days post Jarid2-deletion for Jarid2-/–Jak2V617F/ + and Jarid2+/–Jak2V617F/ + mice respectively, compared to 113-days for Jak2V617F/ + control mice. These data establish Jarid2 as a bona fide tumor suppressor in MPN. To understand the tumor suppressor role of Jarid2, we defined its role in normal hematopoiesis. Transplantation of hematopoietic stem cells (HSCs) or whole bone marrow (WBM) showed a striking difference. Vav-CRE:Jarid2-KO HSCs did not contribute to peripheral blood (PB) chimerism, whereas Vav-CRE:Jarid2-KO WBM contributed to PB lineages at levels comparable to control WBM. These data suggested that a population of cell(s) other than HSCs contributes to hematopoiesis in the absence of Jarid2. Strikingly, Vav-CRE:Jarid2-KO multipotent progenitors (MPPs) engrafted in primary and secondary recipients, generating tri-lineage hematopoiesis effectively. Additionally, MPPs from Jak2V617FJarid2KO mice can propagate MPN, unlike MPPs transplanted from Jak2V617F mice. RNA-seq analysis revealed upregulation of Runx1t1 and Mycn in Jarid2-KO MPPs, genes which can reprogram mature blood cells to a multipotent state, which was associated with depletion of H3K27me3. Expression of Runx1t1 and Mycn in wild-type MPPs endowed long-term repopulating capacity in vivo, phenocopying Jarid2-KO MPPs. Our data suggest that the biological role of Jarid2 in hematopoiesis is to restrict self-renewal capacity in lineage-committed progenitor cells, and that dysregulation of this process can contribute to hematopoietic malignancies.

Abstract 2559: Generation and evaluation of an FLT3 CAR-T cell therapy for the treatment of acute myeloid leukemia

Abstract Background: FLT3 (CD135) is a receptor tyrosine kinase expressed by hematopoietic progenitor cells, and when activated by ligand binding, FLT3 signaling can induce survival, proliferation and differentiation. Acute myeloid leukemia (AML), an indication with high unmet need, derives from hematopoietic progenitor lineage cells; consequently, many AML patient samples retain FLT3 expression on circulating leukemic cells. In comparison to the wide expression seen in AML patient samples, normal tissue expression of FLT3 is restricted to the hematopoietic compartment: a subset of hematopoietic stem cells, a subset of lineage committed progenitor cells and dendritic cells. Cell surface FLT3 protein was not detectable in those solid tissues that express quantifiable FLT3 mRNA (cerebellum, pancreas). As a result of this favorable expression profile, targeting AML cells using T cells expressing a chimeric antigen receptor (CAR) directed against FLT3, is expected to provide benefit to patients. Methods: A panel of fully-human, anti-human FLT3 antibodies were generated, screened for specific FLT3 binding, converted to single chain variable fragments (scFv) and re-evaluated for binding and stability. Three anti-FLT3 scFv sequences were selected for incorporation into CAR constructs. T cells transduced with these CAR constructs were evaluated in vitro for cytotoxicity, proliferation and cytokine secretion, and in vivo in a mouse xenograft model. The lead anti-FLT3 scFv was combined with an anti-CD3 scFv (bispecific T cell engager (BiTE®) format) for evaluation in non-human primates (NHP). Results: Human T cells engineered to express anti-FLT3 CAR constructs were evaluated for cytotoxicity, proliferation and cytokine secretion in the presence of three FLT3-positive cell lines, each with a different number of surface-expressed FLT3 receptors, and one FLT3-negative cell line. While all constructs were selectively active against FLT3-positive cells, one construct was more active than the others against cells expressing very low levels of FLT3; 90% depletion of target cells with ~1600 receptors/cell was observed after 40 hours of co-culture with CAR T cells in a 1:1 ratio. This lead construct was further evaluated in a mouse xenograft model, where FLT3 CAR-T cells provided a survival advantage. The anti-FLT3 scFv used in the lead CAR construct was evaluated in NHP in the form of a BiTE®. In this 16-day, multi-dose study, the FLT3 BiTE® induced elimination of FLT3+ cells as assessed by depletion of FLT3 mRNA-expressing cells (97% reduction in FLT3 mRNA) in the circulation and increased levels of soluble FLT3 ligand in serum. Conclusions: These results demonstrate the potential of using FLT3 CAR-T cell therapy for the treatment of AML. Citation Format: Tara L. Arvedson, Alice Bakker, Herve Lebrec, Gregor Adams, Armen Madiros, Priya Koppikar, Mercedesz Balazs, Mei Gong, Yan Zheng, Rebecca Goldstein, Tony Polverino, Lawren Wu, Angela Coxon. Generation and evaluation of an FLT3 CAR-T cell therapy for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2559.