Phenotype Of Progenitor Cells Research Articles

Glucose intolerance as a consequence of hematopoietic stem cell dysfunction in offspring of obese mice

ObjectiveMaternal obesity is increasingly common and negatively impacts offspring health. Children of mothers with obesity are at higher risk of developing diseases linked to hematopoietic system abnormalities and metabolism such as type 2 diabetes. Interestingly, disease risks are often dependent on the offspring's sex, suggesting sex-specific reprogramming effect of maternal obesity on offspring hematopoietic stem and progenitor cell (HSPC) function. However, the impact of maternal obesity exposure on offspring HSPC function, and the capability of HSPC to regulate offspring metabolic health is largely understudied. This study aims to test the hypothesis that offspring of obese mice exhibit sex-differences in HSPC function that affect offspring's metabolic health. MethodsWe first assessed bone marrow hematopoietic stem and progenitor cell phenotype using postnatal day 21 (P21) and 8-week-old C57BL/6J mice born to control and diet-induced obese dams. We also sorted HSPC (Lineage-, Sca1+, cKit + cells) from P21 mice for competitive primary and secondary transplant, as well as transcriptomic analysis. Body weight, adiposity, insulin tolerance test and glucose tolerance tests were performed in primary and secondary transplant recipient animals. ResultsWe discovered sex-differences in offspring HSPC function in response to maternal obesity exposure, where male offspring of obese dams (MatOb) showed decreased HSPC numbers and engraftment, while female MatOb offspring remained largely unaffected. RNA-seq revealed immune stimulatory pathways in female MatOb offspring. Finally, only recipients of male MatOb offspring HSPC exhibited glucose intolerance. ConclusionsThis study demonstrated the lasting effect of maternal obesity exposure on offspring HSPC function and implicates HSPC in metabolic regulation.

Maternal obesity is associated with metabolic endotoxemia and changes in the phenotype of hematopoietic progenitor cells and monocytes of their offspring at birth

Maternal obesity is associated with metabolic endotoxemia and changes in the phenotype of hematopoietic progenitor cells and monocytes of their offspring at birth

Effects of amyloid-β-mimicking peptide hydrogel matrix on neuronal progenitor cell phenotype

Self-assembling peptide-based hydrogels have become a highly attractive scaffold for three-dimensional (3D) in vitro disease modeling as they provide a way to create tunable matrices that can resemble the extracellular matrix (ECM) of various microenvironments. Alzheimer's disease (AD) is an exceptionally complex neurodegenerative condition; however, our understanding has advanced due to the transition from two-dimensional (2D) to 3D in vitro modeling. Nonetheless, there is a current gap in knowledge regarding the role of amyloid structures, and previously developed models found long-term difficulty in creating an appropriate model involving the ECM and amyloid aggregates. In this report, we propose a multi-component self-assembling peptide-based hydrogel scaffold to mimic the amyloid-beta (β) containing microenvironment. Characterization of the amyloid-β-mimicking hydrogel (Col-HAMA-FF) reveals the formation of β-sheet structures as a result of the self-assembling properties of phenylalanine (Phe, F) through π−π stacking of the residues, thus mimicking the amyloid-β protein nanostructures. We investigated the effect of the amyloid-β-mimicking microenvironment on healthy neuronal progenitor cells (NPCs) compared to a natural-mimicking matrix (Col-HAMA). Our results demonstrated higher levels of neuroinflammation and apoptosis markers when NPCs were cultured in the amyloid-like matrix compared to a natural brain matrix. Here, we provided insights into the impact of amyloid-like structures on NPC phenotypes and behaviors. This foundational work, before progressing to more complex plaque models, provides a promising scaffold for future investigations on AD mechanisms and drug testing. Statement of significanceIn this study, we engineered two multi-component hydrogels: one to mimic the natural extracellular matrix (ECM) of the brain and one to resemble an amyloid-like microenvironment using a self-assembling peptide hydrogel. The self-assembling peptide mimics β-amyloid fibrils seen in amyloid-β protein aggregates. We report on the culture of neuronal progenitor cells within the amyloid-mimicking ECM scaffold to study the impact through marker expressions related to inflammation and DNA damage. This foundational work, before progressing to more complex plaque models, offers a promising scaffold for future investigations on AD mechanisms and drug testing.

Regulation of Oxygen Tension as a Strategy to Control Chondrocytic Phenotype for Cartilage Tissue Engineering and Regeneration.

Cartilage defects and osteoarthritis are health problems which are major burdens on health care systems globally, especially in aging populations. Cartilage is a vulnerable tissue, which generally faces a progressive degenerative process when injured. This makes it the 11th most common cause of global disability. Conservative methods are used to treat the initial phases of the illness, while orthopedic management is the method used for more progressed phases. These include, for instance, arthroscopic shaving, microfracturing and mosaicplasty, and joint replacement as the final treatment. Cell-based implantation methods have also been developed. Despite reports of successful treatments, they often suffer from the non-optimal nature of chondrocyte phenotype in the repair tissue. Thus, improved strategies to control the phenotype of the regenerating cells are needed. Avascular tissue cartilage relies on diffusion for nutrients acquisition and the removal of metabolic waste products. A low oxygen content is also present in cartilage, and the chondrocytes are, in fact, well adapted to it. Therefore, this raises an idea that the regulation of oxygen tension could be a strategy to control the chondrocyte phenotype expression, important in cartilage tissue for regenerative purposes. This narrative review discusses the aspects related to oxygen tension in the metabolism and regulation of articular and growth plate chondrocytes and progenitor cell phenotypes, and the role of some microenvironmental factors as regulators of chondrocytes.

ANIMAL COMPONENT-FREE WORKFLOWS TO DERIVE TENOCYTE-LIKE CELLS FROM HUMAN MESENCHYMAL STROMAL CELLS AND PLURIPOTENT STEM CELLS

Cell-based therapies offer a promising strategy to treat tendon injuries and diseases. Both mesenchymal stromal cells (MSCs) and pluripotent stem cells (PSCs) are good candidates for such applications due to their self-renewing and differentiation capacity. However, the translation of cell-based therapies from bench to bedside can be hindered by the use of animal-derived components in ancillary materials and by the lack of standardised media and protocols for in vitro tenogenic differentiation. To address this, we have optimized animal component-free (ACF) workflows for differentiating human MSCs and PSCs to tenocyte-like cells (TLCs) respectively. MSCs isolated from bone marrow (n = 3) or adipose tissue (n = 3) were expanded using MesenCult™-ACF Plus Culture Kit for at least 2 passages, and differentiated to TLCs in 21 days using a step-wise approach. Briefly, confluent cultures were treated with an ACF tenogenic induction medium for 3 days, followed by treatment with an ACF maturation medium for 18 days. Monolayer cultures were maintained at high density without passaging for the entire duration of the protocol, and the medium was changed every 2 – 3 days. In a similar fashion, embryonic (n = 3) or induced PSCs (n = 3) were first differentiated to acquire a mesenchymal progenitor cell (MPC) phenotype in 21 days using STEMdiff™ Mesenchymal Progenitor Kit, followed by the aforementioned tenogenic protocol for an additional 21 days. In all cases, the optimized workflows using ACF formulations consistently activated a tenogenic transcriptional program, leading to the generation of elongated, spindle-shaped tenomodulin-positive (TNMD+) cells and deposition of an extracellular matrix predominantly composed of collagen type I. In summary, here we describe novel workflows that can robustly generate TLCs from MSCs and hPSC-derived MPCs for potential translational applications.

Recombinant Laminin-511 Fragment (iMatrix-511) Coating Supports Maintenance of Human Nucleus Pulposus Progenitor Cells In Vitro.

The angiopoietin-1 receptor (Tie2) marks specific nucleus pulposus (NP) progenitor cells, shows a rapid decline during aging and intervertebral disc degeneration, and has thus sparked interest in its utilization as a regenerative agent against disc degeneration. However, the challenge of maintaining and expanding these progenitor cells in vitro has been a significant hurdle. In this study, we investigated the potential of laminin-511 to sustain Tie2+ NP progenitor cells in vitro. We isolated cells from human NP tissue (n = 5) and cultured them for 6 days on either standard (Non-coat) or iMatrix-511 (laminin-511 product)-coated (Lami-coat) dishes. We assessed these cells for their proliferative capacity, activation of Erk1/2 and Akt pathways, as well as the expression of cell surface markers such as Tie2, GD2, and CD24. To gauge their regenerative potential, we examined their extracellular matrix (ECM) production capacity (intracellular type II collagen (Col2) and proteoglycans (PG)) and their ability to form spherical colonies within methylcellulose hydrogels. Lami-coat significantly enhanced cell proliferation rates and increased Tie2 expression, resulting in a 7.9-fold increase in Tie2-expressing cell yields. Moreover, the overall proportion of cells positive for Tie2 also increased 2.7-fold. Notably, the Col2 positivity rate was significantly higher on laminin-coated plates (Non-coat: 10.24% (±1.7%) versus Lami-coat: 26.2% (±7.5%), p = 0.010), and the ability to form spherical colonies also showed a significant improvement (Non-coat: 40.7 (±8.8)/1000 cells versus Lami-coat: 70.53 (±18.0)/1000 cells, p = 0.016). These findings demonstrate that Lami-coat enhances the potential of NP cells, as indicated by improved colony formation and proliferative characteristics. This highlights the potential of laminin-coating in maintaining the NP progenitor cell phenotype in culture, thereby supporting their translation into prospective clinical cell-transplantation products.

Aldehyde Dehydrogenase, a Marker of Normal and Malignant Stem Cells, Typifies Mesenchymal Progenitors in Perivascular Niches.

Innate mesenchymal stem cells exhibiting multilineage differentiation and tissue (re)generative-or pathogenic-properties reside in perivascular niches. Subsets of these progenitors are committed to either osteo-, adipo-, or fibrogenesis, suggesting the existence of a developmental organization in blood vessel walls. We evaluated herein the activity of aldehyde dehydrogenase, a family of enzymes catalyzing the oxidation of aldehydes into carboxylic acids and a reported biomarker of normal and malignant stem cells, within human adipose tissue perivascular areas. A progression of ALDHLow to ALDHHigh CD34+ cells was identified in the tunica adventitia. Mesenchymal stem cell potential was confined to ALDHHigh cells, as assessed by proliferation and multilineage differentiation in vitro of cells sorted by flow cytometry with a fluorescent ALDH substrate. RNA sequencing confirmed and validated that ALDHHigh cells have a progenitor cell phenotype and provided evidence that the main isoform in this fraction is ALDH1A1, which was confirmed by immunohistochemistry. This demonstrates that ALDH activity, which marks hematopoietic progenitors and stem cells in diverse malignant tumors, also typifies native, blood vessel resident mesenchymal stem cells.

Development of Brain-Derived Bioscaffolds for Neural Progenitor Cell Culture.

Biomaterials derived from brain extracellular matrix (ECM) have the potential to promote neural tissue regeneration by providing instructive cues that can direct cell survival, proliferation, and differentiation. This study focused on the development and characterization of microcarriers derived from decellularized brain tissue (DBT) as a platform for neural progenitor cell culture. First, a novel detergent-free decellularization protocol was established that effectively reduced the cellular content of porcine and rat brains, with a >97% decrease in the dsDNA content, while preserving collagens (COLs) and glycosaminoglycans (GAGs). Next, electrospraying methods were applied to generate ECM-derived microcarriers incorporating the porcine DBT that were stable without chemical cross-linking, along with control microcarriers fabricated from commercially sourced bovine tendon COL. The DBT microcarriers were structurally and biomechanically similar to the COL microcarriers, but compositionally distinct, containing a broader range of COL types and higher sulfated GAG content. Finally, we compared the growth, phenotype, and neurotrophic factor gene expression levels of rat brain-derived progenitor cells (BDPCs) cultured on the DBT or COL microcarriers within spinner flask bioreactors over 2 weeks. Both microcarrier types supported BDPC attachment and expansion, with immunofluorescence staining results suggesting that the culture conditions promoted BDPC differentiation toward the oligodendrocyte lineage, which may be favorable for cell therapies targeting remyelination. Overall, our findings support the further investigation of the ECM-derived microcarriers as a platform for neural cell derivation for applications in regenerative medicine.

Nitric oxide promotes cell-matrix adhesion of endothelial progenitor cells under hypoxia condition via ITGA5 CpG promoter demethylation

Hypoxia or low oxygen tension causes changes in the structure and functional phenotype of the endothelial progenitor cells (EPCs). EPCs are found to be involved in angiogenesis and vascular repair. However, EPC's role in cell-matrix adhesion under hypoxia conditions is not clearly established. Nitric oxide (NO) exerts a wide range of biological functions, especially in regulating the mobilization and vascular repair of EPCs. In contrast, the link between NO and its role in cell-matrix deadhesion under hypoxia is not studied yet. Here, we investigated the protective role of NO in hypoxia-induced cell-matrix deadhesion of EPCs through an epigenetic mechanism. The EPCs were exposed to 2% hypoxia in the presence or absence of 10 μM Spermine NONOate (NO donor). The result demonstrates that hypoxia exposure intensified mitochondrial oxidative damage and energy defects. Using miScript miRNA qPCR array-based screening, the study found miR-148 as a novel target of hypoxia-induced DNMT1 activation. Mechanistically, the study discovered that hypoxia suppressed miR-148 levels and stimulated EPCs cell-matrix deadhesion via increasing DNMT1 mediated Integrin alpha-5 (ITGA5) CpG promoter hypermethylation. Treatment with a mitochondria-targeted antioxidant, MitoTEMPO, or epigenetic DNMT inhibitor, 5′-azacitidine, or miR-148 overexpression in hypoxic EPCs culture, prevented the cell-matrix deadhesion compared to hypoxic EPCs. Further, treatment of spNO or transient expression of eNOS-GFP attenuated hypoxia-induced cell-matrix deadhesion via inhibition of ITGA5 CpG island promoter methylation. In conclusion, the study provides evidence that NO is essential for cell-matrix adhesion of EPCs by epigenetically mitigating ITGA5 CpG promoter hypermethylation under hypoxia conditions. This finding uncovers the previously undefined mechanism of NO-mediated diminution of hypoxia-induced cell-matrix deadhesion and dysfunction induced by low oxygen tension.

Hematologic dysfunction in cancer: Mechanisms, effects on antitumor immunity, and roles in disease progression.

With the major advances in cancer immunology and immunotherapy, it is critical to consider that most immune cells are short-lived and need to be continuously replenished from hematopoietic stem and progenitor cells. Hematologic abnormalities are prevalent in cancer patients, and many ground-breaking studies over the past decade provide insights into their underlying cellular and molecular mechanisms. Such studies demonstrate that the dysfunction of hematopoiesis is more than a side-effect of cancer pathology, but an important systemic feature of cancer disease. Here we review these many advances, covering the cancer-associated phenotypes of hematopoietic stem and progenitor cells, the dysfunction of myelopoiesis and erythropoiesis, the importance of extramedullary hematopoiesis in cancer disease, and the developmental origins of tumor associated macrophages. We address the roles of many secreted mediators, signaling pathways, and transcriptional and epigenetic mechanisms that mediate such hematopoietic dysfunction. Furthermore, we discuss the important contribution of the hematopoietic dysfunction to cancer immunosuppression, the possible avenues for therapeutic intervention, and highlight the unanswered questions and directions for future work. Overall, hematopoietic dysfunction is established as an active component of the cancer disease mechanisms and an important target for therapeutic intervention.

Accurate Estimation of Single-Cell Differentiation Potency Based on Network Topology and Gene Ontology Information.

One important task in single-cell analysis is to quantify the differentiation potential of single cells. Though various single-cell potency measures have been proposed, they are based on individual biological sources, thus not robust and reliable. It is still a challenge to combine multiple sources to generate a relatively reliable and robust measure to estimate differentiation. In this paper, we propose a New Centrality measure with Gene ontology information (NCG) to estimate single-cell potency. NCG is designed by combining network topology property with edge clustering coefficient, and gene function information using gene ontology function similarity scores. NCG distinguishes pluripotent cells from non-pluripotent cells with high accuracy, correctly ranks different cell types by their differentiation potency, tracks changes during the differentiation process, and constructs the lineage trajectory from human myoblasts into skeletal muscle cells. These indicate that NCG is a reliable and robust measure to estimate single-cell potency. NCG is anticipated to be a useful tool for identifying novel stem or progenitor cell phenotypes from single-cell RNA-Seq data. The source codes and datasets are available at https://github.com/Xinzhe-Ni/NCG.

Receptor tyrosine kinase inhibitors negatively impact on pro-reparative characteristics of human cardiac progenitor cells

Receptor tyrosine kinase inhibitors improve cancer survival but their cardiotoxicity requires investigation. We investigated these inhibitors’ effects on human cardiac progenitor cells in vitro and rat heart in vivo. We applied imatinib, sunitinib or sorafenib to human cardiac progenitor cells, assessing cell viability, proliferation, stemness, differentiation, growth factor production and second messengers. Alongside, sunitinib effects were assessed in vivo. Inhibitors decreased (p < 0.05) cell viability, at levels equivalent to ‘peak’ (24 h; imatinib: 91.5 ± 0.9%; sunitinib: 83.9 ± 1.8%; sorafenib: 75.0 ± 1.6%) and ‘trough’ (7 days; imatinib: 62.3 ± 6.2%; sunitinib: 86.2 ± 3.5%) clinical plasma levels, compared to control (100% viability). Reduced (p < 0.05) cell cycle activity was seen with imatinib (29.3 ± 4.3% cells in S/G2/M-phases; 50.3 ± 5.1% in control). Expression of PECAM-1, Nkx2.5, Wnt2, linked with cell differentiation, were decreased (p < 0.05) 2, 2 and 6-fold, respectively. Expression of HGF, p38 and Akt1 in cells was reduced (p < 0.05) by sunitinib. Second messenger (p38 and Akt1) blockade affected progenitor cell phenotype, reducing c-kit and growth factor (HGF, EGF) expression. Sunitinib for 9 days (40 mg/kg, i.p.) in adult rats reduced (p < 0.05) cardiac ejection fraction (68 ± 2% vs. baseline (83 ± 1%) and control (84 ± 4%)) and reduced progenitor cell numbers. Receptor tyrosine kinase inhibitors reduce cardiac progenitor cell survival, proliferation, differentiation and reparative growth factor expression.

Abstract P010: Antigen dominance hierarchies shape CD8 T cell phenotypes and immunotherapy response in tumors

Abstract CD8 T cell responses against different tumor neoantigens occur simultaneously, yet it is unclear whether they interact to potentiate or antagonize the overall anti-tumor response. In a genetically engineered mouse model of lung adenocarcinoma, we find that antigen dominance hierarchies are established in tumors wherein the antigen that most stably binds MHC dominates the CD8 T cell response. This negatively impacts the response to subdominant antigens, suppressing T cell expansion, differentiation and effector function; a phenotype that is reversed when the dominant antigen is removed. Intriguingly, the subdominant response is also enriched for a TCF1+ progenitor cell phenotype that has been correlated with response to immune checkpoint blockade (ICB) therapy. However, we find that the subdominant response does not preferentially benefit from ICB due to predominance of a dysfunctional subset of TCF1+ cells marked by CCR6 expression and differentiation to a Tc17 phenotype. CCR6+ TCF1+ cells are also found broadly across human cancers and do not correlate with patient response to ICB. This subset appears to be derived from poor T cell receptor stimulation, due to competition of T cells for good interactions with antigen presenting cells. Vaccination eliminates CCR6+ TCF1+ cells and disrupts the antigen dominance hierarchy, preferentially expanding the subdominant CD8 T cell response in tumors. Overall enrichment of TCF1+ cells is maintained amongst the subdominant response post-vaccination and current studies are exploring whether this promotes more durable tumor control or better response to ICB. These findings provide strong rationale for evaluating the relative response to high versus low pMHC stability antigens in clinical trials of pooled neoantigen vaccines, where low stability, subdominant antigens may contribute more to tumor control than previously realized. Citation Format: Megan L. Burger, Amanda M. Cruz, Grace E. Crossland, Giorgio Gaglia, Cecily C. Ritch, Sarah E. Blatt, Arjun Bhutkar, Sara Z. Tavana, Sandro Santagata, Tyler Jacks. Antigen dominance hierarchies shape CD8 T cell phenotypes and immunotherapy response in tumors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P010.

Isolation and phenotyping of cardiac-derived progenitor cells from neonatal mice

Dysfunctions of resident progenitor cells play a significant role in the pathogenesis of decreased myocardial contractility in heart failure, so the most promising approaches for the treatment of heart disease are cardiac-derived stem/progenitor cells (CSCs). Materials and methods. Protocols for progenitor cell cultures from different parts of the heart of newborn FVB/N mice have been developed and their proliferative potential has been characterized. Comparative analysis of the expression of CD31, CD34, CD44, CD45, CD73, CD90, CD105, CD117, CD309 and troponin I by cells from native myocardial biopsies and in the obtained cultures was performed by flow cytometric immunophenotyping. Results. The expression of mesenchymal markers CD44 and CD90 in the absence of the hematopoietic marker CD45 was demonstrated in early passages in mouse myocardial progenitor cell cultures. Relatively high expression of CD34 and CD31 was found. The presence of a minor population of CD44+117+ cells which correspond to the phenotype of cardiac progenitor cells, was detected. Expression of troponin I as one of the key markers of cardiomyocytes as well as the vascular endothelial growth factor receptor has been confirmed in terminally differentiated cultures of cells with contractile activity. Conclusions. It was found that newborn mice in the myocardial tissue contain more cells with the expression of markers of cardiac progenitors than in adult animals. The relative content of such cells is higher in the atria than in the ventricles. Cardiac progenitor cells in neonatal mice derived from the atrial appendages have better proliferative potential than cell cultures isolated from the ventricles.

Treating Metastatic Brain Cancers With Stem Cells.

Stem cell therapy may present an effective treatment for metastatic brain cancer and glioblastoma. Here we posit the critical role of a leaky blood-brain barrier (BBB) as a key element for the development of brain metastases, specifically melanoma. By reviewing the immunological and inflammatory responses associated with BBB damage secondary to tumoral activity, we identify the involvement of this pathological process in the growth and formation of metastatic brain cancers. Likewise, we evaluate the hypothesis of regenerating impaired endothelial cells of the BBB and alleviating the damaged neurovascular unit to attenuate brain metastasis, using the endothelial progenitor cell (EPC) phenotype of bone marrow-derived mesenchymal stem cells. Specifically, there is a need to evaluate the efficacy for stem cell therapy to repair disruptions in the BBB and reduce inflammation in the brain, thereby causing attenuation of metastatic brain cancers. To establish the viability of stem cell therapy for the prevention and treatment of metastatic brain tumors, it is crucial to demonstrate BBB repair through augmentation of vasculogenesis and angiogenesis. BBB disruption is strongly linked to metastatic melanoma, worsens neuroinflammation during metastasis, and negatively influences the prognosis of metastatic brain cancer. Using stem cell therapy to interrupt inflammation secondary to this leaky BBB represents a paradigm-shifting approach for brain cancer treatment. In this review article, we critically assess the advantages and disadvantages of using stem cell therapy for brain metastases and glioblastoma.

Chromatin Accessibility Analysis Reveals Epigenetic Evolution Is a Common Mechanism of Relapse in Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is associated with a poor prognosis and high rates of relapse despite aggressive treatments including high dose chemotherapy. To understand the clonal dynamics and genetic evolution of relapsed AML, we analyzed a cohort of 142 previously published genotyped and paired diagnosis-relapse AML samples. 40% of queried cases exhibited no major changes in somatic mutations upon relapse, and genetically stable clonal structure correlated with increased relapse probability. Thus, we hypothesized epigenetic reprogramming plays a role in these cases and AML relapse in general. Here, we examine the epigenetic landscape of relapsed AML and characterize the cis and trans regulatory elements that correlate with AML relapse in the presence or absence of genetic evolution.Methods: We identified 27 viable cryopreserved paired diagnosis and relapse samples from the same patient treated at Stanford with high-dose chemotherapy. Leukemic blasts and (when possible) leukemia stem cell (LSC) enriched populations were FACS purified and prepared for genotyping with a myeloid malignancy targeted sequencing panel as well as ATAC-seq for chromatin accessibility profiling. Single-cell ATAC-seq was further performed on select samples to investigate regulatory reprogramming in cell subpopulations. We then performed integrative analysis to uncover the interplay between genetic lesions, epigenetic regulatory programs, and gene accessibility in relapsed AML.Results: Genotyping analysis of these AML specimens revealed that 40% of samples exhibited no changes in AML-related genetic alterations upon relapse (hereby referred to as “stable” samples). Chromatin accessibility analysis revealed these stable samples had a distinct epigenetic signature, modulating similar gene accessibility programs and sharing enhancer loci that become accessible across all stable relapse samples. These sequences included genes involved in chromatin organization and compaction, as well as those involved in transcriptional control of hematopoietic differentiation and myeloid cell maturation. We also observed several regulatory signatures present at relapse specific for AML subtypes, including NPM1/FLT3 double mutant AML and those with mutations in transcription factors such as CEBPA or RUNX1.We then performed single-cell ATAC-seq on genetically stable samples to further characterize the sub-clonal epigenetic dynamics between the diagnosis and relapse cells. Several samples exhibited regulatory heterogeneity in multiple cell subpopulations that changed significantly at relapse. One subpopulation of interest was characterized by increased GATA and RUNX family transcription factor motif accessibility at relapse, indicating a shift toward a less differentiated progenitor cell phenotype. In addition, we identified a subpopulation of cells at diagnosis that were epigenetically similar to the major epigenetic states present at relapse, indicating that selection for specific epigenetic subclones may occur in AML patients during therapy in the absence of additional genetic lesions.Finally, given the critical role of LSCs in AML pathogenesis and their possible role as a reservoir for AML relapse, we analyzed LSC-enriched subpopulations in a subset of our cohort. ATAC-seq analysis indicated these cells shared several LSC-specific epigenetic features between samples, are distinguished from leukemia blasts by distinct regulatory programs, and undergo epigenetic remodeling between initial diagnosis and relapse. Gene accessibility analysis also revealed a shared LSC gene expression signature that also shifted at relapse. These data indicate a specific, distinct epigenomic signature for LSC enriched cell populations, and that epigenetic evolution at relapse occurs intracellularly, rather than reflecting heterogeneity in cellular subpopulations upon AML relapse.Conclusion: This study reveals that epigenetic remodeling in the absence of genetic evolution is a mechanism through which AML relapse occurs. We show that chromatin reorganization of genes and regulatory sequences occurs in these AML cells, leading to a permissive cell state that might be resistant to conventional treatment. Ongoing work includes dissecting the subclonal structure of these AML cells and identifying the relationship between gene regulatory networks that contribute to relapse. DisclosuresEdiriwickrema: Nanosive SAS: Patents & Royalties. Majeti: BeyondSpring Inc.: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc..

Colony Forming Potential and Protein Composition of Commercial Umbilical Cord Allograft Products in Comparison With Autologous Orthobiologics

Background: Umbilical cord (UC) connective tissues contain plastic-adherent, colony forming unit–fibroblasts (CFU-Fs) amenable to culture expansion for potential therapeutic use. Recently, UC-derived allograft products have been made available to practitioners in orthopaedics and other specialties, by companies purporting “stem cell”–based healing. However, such marketing claims conflict with existing regulations for these human tissues, generating questions over the cellular and protein composition of current commercially available UC allograft products. Purpose: To evaluate commercial UC allograft products for viable cells, CFU-Fs, and protein makeup. Study Design: Descriptive laboratory study. Methods: Five commercial UC allograft products claiming to contain viable, undescribed “stem cells,” 2 obtained from UC blood (UCB) and 3 from UC tissue (UCT), were analyzed. Image-based methods were used to measure cell concentration and viability, a traditional CFU-F assay was used to evaluate in vitro behavior indicative of a connective tissue progenitor cell phenotype often referred to as mesenchymal stem/stromal cells, and quantitative immunoassay arrays were used to measure a combination of cytokines and growth factors. Bone marrow concentrate (BMC) and plasma derived from the blood and bone marrow of middle-aged individuals served as comparative controls for cell culture and protein analyses, respectively. Results: Viable cells were identified within all 5 UC allograft products, with those derived from UCB having greater percentages of living cells (40%-59%) than those from UCT (1%-22%). Compared with autologous BMC (>95% viability and >300 million living cells), no CFU-Fs were observed within any UC allograft product (<15 million living cells). Moreover, a substantial number of proteins, particularly those within UCB allograft products, were undetectable or present at lower concentrations compared with blood and bone marrow plasma controls. Interestingly, several important growth factors and cytokines, including basic fibroblast growth factor, hepatocyte growth factor, interleukin-1 receptor antagonist, and osteoprotegerin, were most prevalent in 1 or more UCT allograft products as compared with blood and bone marrow plasma. Conclusion: CFU-Fs, often referred to as stem cells, were not found within any of the commercial UC allograft products analyzed, and clinicians should remain wary of marketing claims stating otherwise. Clinical Relevance: Any therapeutic benefit of current UC allograft products in orthopaedic medicine is more likely to be attributed to their protein composition (UCT > UCB) or inclusion of cells without colony forming potential (UCB > UCT).

AML1/ETO and FLT3-ITD Cooperate to Induce Acute Myeloid Leukemia in Mice

Acute myeloid leukemia (AML), the most common form of acute leukemia in adults, accounts for more than 40% of all leukemia deaths. The AML1-ETO (A) fusion gene resulting from t(8;21) is a recurring genetic alteration in human AML, typically associated with a favorable outcome. Internal tandem duplications (ITD) in the juxtamembrane domain of Fms-like tyrosine kinase 3 (Flt3) result in constitutive ligand-independent signaling. Flt3ITD is one of the most frequently identified molecular abnormalities in AML, and when present, portends unfavorable clinical outcomes. AML1/ETO and FLT3ITD can co-exist in human AML, indicating that these lesions cooperate to promote disease development. Neither AML1/ETO nor Flt3ITD alone are sufficient to induce AML in mice. To determine whether coordinated expression of AML1/ETO and Flt3ITD would cause AML, Mx1-Cre x LSL AML1-ETO/+ x FLT3ITD/ITD (MAFF) mice were generated. In this system, an Mx1 promoter-driven Cre recombinase removes a lox-stop-lox (LSL) cassette upstream of a knock-in human AML1/ETO fusion gene in hematopoietic progenitor cells, while FLT3ITD is constitutively expressed. MAFF mice developed AML at 4-5 weeks of age with 100% penetrance. The AML was associated with severe leukocytosis, splenomegaly and dense bone marrow infiltration by immature-appearing blasts, and was universally lethal. Flow cytometry of bone marrow from MAFF mice revealed a striking expansion of cells harboring a hematopoietic stem cell (HSC) and multipotent progenitor (MP) cell phenotype, indicative of a block in normal myeloid differentiation. No lymphoid leukemias were observed. Interestingly, two copies of the FLT3ITD allele were required for AML to develop in this model. Transplantation of as few as 105 bone marrow cells from primary MAFF mice into lethally-irradiated secondary recipients resulted in 100% engraftment and lethality, with a leukemia identical to that which developed in primary MAFF mice, albeit with delayed latency. Mixed bone marrow chimeras were generated using MAFF marrow along with marrow from syngeneic, wild-type C57BL/6 or Rag2-/-γC-/- mice (lacking mature B, T and NK cells). Chimeras generated with MAFF and Rag2-/-γC-/- marrow succumbed more rapidly than those with MAFF and wild-type marrow, indicating that adaptive immunity and/or NK cells provided some level of active immune surveillance against MAFF AML. MAFF mice were crossed with Rosa26-LSLSIY/+ mice (referred to as MAFFS mice) in order to incorporate inducible expression of a tractable peptide antigen (SIYRYYGL; SIY) in leukemia cells. Through the use of mixed bone marrow chimeric approaches, we are now capable of monitoring SIY antigen-specific CD8+ T cell responses in MAFFS AML-bearing animals, including contexts in which the SIY antigen is shared (leukemia-associated antigen) or expressed only by leukemia cells (leukemia-specific antigen). In conclusion, we have generated the first autochthonous murine AML model in which the role of host immune surveillance can be interrogated. We expect that the MAFF AML model will also be useful for studies regarding leukemia biology and treatment. DisclosuresNo relevant conflicts of interest to declare.

British Society for Haematology guidelines for the diagnosis and evaluation of prognosis of Adult Myelodysplastic Syndromes.

British Society for Haematology guidelines for the diagnosis and evaluation of prognosis of Adult Myelodysplastic Syndromes.

Study of inherited thrombocytopenia resulting from mutations in ETV6 or RUNX1 using a human pluripotent stem cell model.

SummaryInherited thrombocytopenia results in low platelet counts and increased bleeding. Subsets of these patients have monoallelic germline mutations in ETV6 or RUNX1 and a heightened risk of developing hematologic malignancies. Utilizing CRISPR-Cas9, we compared the in vitro phenotype of hematopoietic progenitor cells and megakaryocytes derived from induced pluripotent stem cell (iPSC) lines harboring mutations in either ETV6 or RUNX1. Both mutant lines display phenotypes consistent with a platelet-bleeding disorder. Surprisingly, these cellular phenotypes were largely distinct. The ETV6-mutant iPSCs yield more hematopoietic progenitor cells and megakaryocytes, but the megakaryocytes are immature and less responsive to agonist stimulation. On the contrary, RUNX1-mutant iPSCs yield fewer hematopoietic progenitor cells and megakaryocytes, but the megakaryocytes are more responsive to agonist stimulation. However, both mutant iPSC lines display defects in proplatelet formation. Our work highlights that, while patients harboring germline ETV6 or RUNX1 mutations have similar clinical phenotypes, the molecular mechanisms may be distinct.