Activity Of Progenitor Cells Research Articles

Interleukin-6 upregulates extracellular matrix gene expression and transforming growth factor β1 activity of tendon progenitor cells

BackgroundProlonged inflammation during tendon healing and poor intrinsic healing capacity of tendon are causal factors associated with tendon structural and functional degeneration. Tendon cells, consisting of mature tenocytes and tendon progenitor cells (TPC) function to maintain tendon structure via extracellular matrix (ECM) synthesis. Tendon cells can succumb to tissue cytokine/chemokine alterations during healing and consequently contribute to tendon degeneration. Interleukin-(IL-)1β, IL-6 and TNFα are key cytokines upregulated in injured tendons; the specific effects of IL-6 on flexor tendon-derived TPC have not been discerned.MethodsPassage 3 equine superficial digital flexor tendon (SDFT)-derived TPC were isolated from 6 horses. IL-6 impact on the viability (MMT assay with 0, 1, 5 and 10 ng/mL concentrations), migration (scratch motility assay at 0, 10ng/mL concentration) of TPC in monolayer culture were assessed. IL-6 effect on tendon ECM and chondrogenic gene expression (qRT-PCR), TGFβ1 gene expression and activity (ELISA), and MMP-1, -3 and − 13 gene expression of TPC was evaluated.ResultsIL-6 decreased TPC viability and migration. IL-6 treatment at 10 ng/mL significantly up-regulated TGFβ1 gene expression (6.3-fold; p = 0.01) in TPC, and significantly increased the TGFβ1 concentration in cell culture supernates. IL-6 (at 10 ng/mL) significantly up-regulated both tendon ECM (COL1A1:5.3-fold, COL3A1:5.4-fold, COMP 5.5-fold) and chondrogenic (COL2A1:3.9-fold, ACAN:6.2-fold, SOX9:4.8-fold) mRNA expression in TPC. Addition of SB431542, a TGFβ1 receptor inhibitor, to TPC in the presence of IL-6, attenuated the up-regulated tendon ECM and chondrogenic genes.ConclusionIL-6 alters TPC phenotype during in vitro monolayer culture. Pro- and anti-inflammatory roles of IL-6 have been implicated on tendon healing. Our findings demonstrate that IL-6 induces TGFβ1 activity in TPC and affects the basal TPC phenotype (as evidenced via increased tendon ECM and chondrogenic gene expressions). Further investigation of this biological link may serve as a foundation for therapeutic strategies that modulate IL-6 to enhance tendon healing.

TNFα induces Caspase-3 activity in hematopoietic progenitor cells CD34+, CD33+, and CD41 + of myelodysplastic syndromes

BackgroundCytopenia is the primary feature of Myelodysplastic Syndrome, even in the presence of hypercellular bone marrow. TNFα is recognized as both a proinflammatory, and proapoptotic cytokine with a well established role in promoting apoptosis in MDS. Therefore, TNFα has the potential to be a valuable biomarker for predicting the progression of cytopenia in MDS. This study aims to establish the role of TNFα exposure in triggering apoptosis through caspase-3 activity in CD34+, CD33+, and CD41 + cells in MDS.MethodsThis study is an in vitro comparative experimental research. Bone marrow mononuclear cells were isolated as the source of hematopoietic progenitor cells. Subsequently, CD34+, CD33+, and CD41 + cells were exposed to rhTNFα, and the caspase-3 activity was measured using flowcytometry.ResultsIn MDS CD33 + and CD41 + caspase-3 activity of rhTNFα exposed cells was significantly higher than without exposed cells. The opposite result was found in CD34 + cells, where the caspase-3 activity without rhTNFα exposed cells was significantly higher than rhTNFα exposed cells.ConclusionrhTNFα exposure led to an elevation in caspase-3 activity in MDS progenitor cells, especially in those that had differentiated into myeloid cell CD33 + and megakaryocyte cell CD41+, as opposed to the early progenitor cells CD34+.

Double-Negative T Cells Attenuate Acute Graft-Versus-Host Disease By Modulating CD4 + T Cell Activation Post-Allo-HSCT

Background: Acute graft-versus-host disease (aGVHD) is a major and life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Double-negative T cells (DNTs) have demonstrated potent cytotoxicity against various hematological tumors, and emerging evidence from animal experiments and clinical studies suggests their potential in predicting and treating aGVHD. Thus, DNTs may be a novel and versatile immunotherapy for aGVHD and relapse management post-allo-HSCT due to the function of harnessing the anti-leukemic effects and immune regulation. Nevertheless, the precise mechanisms underlying DNT-mediated aGVHD prevention remain unclear. Results: In vitro, DNTs inhibited the proliferation of CD4 + T cells in a dose-dependent manner, and significantly inhibited the expression of CD25 on CD4 + T cells. CD4 + T cells inhibited by DNTs displayed decreased interleukin-2 secretion, reduced cell volume, nuclear condensation, and diminished mitochondrial number and volume. Transcriptome sequencing analysis verified significant downregulation of DNA replication, cell cycle, mitochondrial matrix metabolism, JAK-STAT, interleukin-17, and mitosis signaling pathways in CD4 + T cells co-cultured with DNTs. In addition, we found DNTs arrested CD4 + T cells at the G0/G1 phase, hindering their transition to the S phase, and inhibited CD4 + T cell proliferation by the CD25-JAK-STAT pathway. However, DNTs had no adverse effects on the activity and colony-forming capacity of allogeneic hematopoietic progenitor cells in vitro. Importantly, DNT infusion significantly improved the survival of mice in aGVHD model, reduced splenomegaly, downregulated CD25 expression on CD4 + T cells, and attenuated CD4 + T cell infiltration into host lungs, liver, and spleen, and effectively ameliorated organ damage (Figure 1). Conclusion: Our findings suggest that DNTs attenuate acute aGVHD by affecting the proliferation and activation of CD4 +T cells via the CD25-JAK-STAT pathway, which is important to the prevention and management of aGVHD.

Role of Lama4 Expression in Bone Marrow Niche for the Progression and Treatment Response of Chronic Myeloid Leukemia

Although tyrosine kinase inhibitors (TKIs) have dramatically improved the treatment outcomes of chronic myeloid leukemia (CML), TKIs cannot eliminate CML-initiating stem cells (LSCs), leading to CML persistence and relapse post TKI-discontinuation. Thus, it is imperative to develop more effective therapeutic options to overcome the treatment challenges. Targeting the bone marrow (BM) niche has become one of the research focuses in drug discovery since increasing evidence suggests that BM niches protect CML LSCs. Laminin alpha4 chain (Lama4), a functional chain for several laminin isoforms, is widely expressed in bone marrow (BM). We have recently shown the critical impact of LAMA4 expression in the microenvironment for hematopoiesis regeneration including megakaryocyte maturation and acute myeloid leukemia progression in mice (Cai et al., Blood, 2022). Furthermore, we have by RNA sequencing shown downregulation of LAMA4 mRNA in freshly isolated BM mesenchymal stem cells (MSCs) from newly diagnosed patients with CML (Dolinska, Cai, Mansson, et al., Blood, 2023). However, the functional impact of the LAMA4 expression on CML LSCs remains to be investigated. We have here explored the role of LAMA4 in CML development and drug response by using both mouse models and primary patient BM samples. To first assess the in vivo impact of LAMA4 expression in BM niches on CML development, we transplanted BM cells (CD45.2) from Scl- tTA× TRE-BCR-ABL1 (BCR-ABL1) mice prior or following tetracycline withdrawal into irradiated Lama4+/+ and Lama4-/- CD45.1mice and analyzed CML progression and BCR-ABL1 LSC activity by flow cytometry and droplet digital PCR (ddPCR). Following transplantation of unfractionated BM cells from the BCR-ABL1 mice with CML induced by tetracycline withdrawal, Lama4-/- recipients showed significantly increased platelet counts in blood compared to Lama4+/+ recipients at 2-8 weeks post-transplantation (P=0.005 at week 2, P= 0.008, n=7-8, at week 8), which is in contrast to the impaired platelet recovery in non-transplanted Lama4-/- mice following sublethal irradiation. This suggests that Lama4 deficient microenvironment is favorable for platelet production from BCR-ABL1 CML cells. Consistent with this, megakaryocyte-erythrocyte progenitors (MEPs) derived from BCR-ABL1+ cells were significantly increased in Lama4 -/- BM at the endpoint (8 weeks, p=0.02, n=3). Megakaryocyte progenitors (MkPs) seemed to be increased in Lama4-/- recipient BM (P=0.05, n=3). Intriguingly, when LIN -SCA1 +KIT + CD150 + hematopoietic stem cells from BCR-ABL1 mice without prior tetracycline withdrawal were transplanted, the donor-derived MEPs were significantly reduced in Lama4-/- BM (P=0.02, n=4-6), which is accompanied with a trend of reduction in MkP but no changes in platelets. ddPCR analysis indicated that the ratio of BCR-ABL1 + cell fraction to total CD45.2 + cells (detected by FACS) seemed to be lower in Lama4-/- recipients (33.6%) than that in Lama4+/+ recipients (41.5%), raising a possibility that Lama4-/- niches is less suitable for maintenance of BCR-ABL1 + cells. Together, these data point to a possible differential regulation of CML stem and progenitor cells by the Lama4 expression. More work will be done to dissect the regulatory mechanisms through in vitro co-cultures, transplantation and confocal imaging. In parallel, the functional impact of LAMA4 on human CML LSC survival was evaluated by a revised long-term culture-initiating cell assay using CML patient derived MSCs. The preliminary result indicated that recombinant LAMA4 peptides appeared to inhibit CML patient BM-derived CD34 +CD38 - cell growth. In conclusion, LAMA4 is downregulated in BM MSCs and endothelial cells of CML patients at diagnosis. Lama4-/- microenvironment might act differentially in regulating BCR-ABL1 + hematopoietic stem and progenitor cell activity post-transplantation. However, more work is required to validate the finding and the effect of LAMA4 peptides on the megakaryocyte differentiation and maturation.

Expression of Ki67 and CD68+ cells of red bone marrow monocyte sprout under triptorelin administration in the hypothalamic-pituitary-testis regulatory system: the experimental study

Background. Red bone marrow (RBM) is the main organ of human haemopoiesis. Monocytopoiesis plays an important role in the formation of transitional states: from normal to pathology and in the transformation of pathological processes from one stage to another. In modern urological practice, the substance triptorelin is widely used for androgen deprivation therapy, according to the recommendations of the European Association of Urology. Ki67, a commercially available monoclonal antibody that reacts with a nuclear antigen detected only in proliferating cells, is used to assess immunohistochemical changes. CD68 is a valuable cytochemical marker for immunostaining of monocytes/macrophages during histochemical analysis of tissues in inflammation, cancer and other immunohistopathological purposes. The purpose of the study is to evaluate the proliferative activity and differentiation of progenitor cells through the expression of Ki67 and CD68+ monocyte sprouting of RBM under chemical castration of central origin in male rats caused by the administration of triptorelin solution with quercetin addition to the diet for one year. Materials and methods. The study was conducted on 60 adult male white rats. They were divided into 3 groups: group I — control (n = 10), group II (n = 25) — subcutaneous injection of triptorelin, group III (n = 25) — subcutaneous injection of triptorelin acetate and quercetin. Immunohistochemical analysis of biopsy specimens was conducted following a standard protocol at the Department of Pathological Anatomy in Sumy State University, under the supervision of the Head of the Department, Prof. Romaniuk A.M. Results. The study evaluated Ki67 expression on microsections of rat red bone marrow through immunohistochemistry, which exclusively reacted with nuclear antigen in the monocyte sprout’s proliferating cells. Irregular changes were revealed depending on the experimental groups and time periods. Immunohistochemical analysis of RBM tissue using anti-CD68 antibodies in the experimental groups revealed a strong positive cytoplasmic response in monocytes and resident macrophages located in the monocyte sprout and surrounding environment. The data of the two experimental groups of RBM had a noticeable proliferating compartment, as evidenced by the high content of mitotically active DNA in them. These data correspond to the results obtained in the experiment with triptorelin, where we found a marked positivity of Ki67, depending on the timing of the experiment and the addition of the flavonoid quercetin. This discrepancy suggests that bone marrow cells that grow and proliferate under normal conditions are guided by natural control mechanisms and may lose their Ki67 expression after leaving the progenitor compartment and entering the differentiation compartment. Conclusions. Triptorelin administration induces hormonal imbalance in the hypothalamus-pituitary-testis-RBM system, resulting in quantitative and qualitative alterations in the cells of the RBM monocytic lineage. The level of cell proliferation, as measured by Ki67, is highest during the third month of observation. Cytoplasmic expression of CD68 is evident in two experimental groups from the third to the sixth month, suggesting activation of immunoreactive cells as they migrate from the progenitor compartment to the differentiation compartment.

MTOR inhibition abrogates human mammary stem cells and early breast cancer progression markers

BackgroundMammary physiology is distinguished in containing adult stem/progenitor cells that are actively amending the breast tissue throughout the reproductive lifespan of women. Despite their importance in both mammary gland development, physiological maintenance, and reproduction, the exact role of mammary stem/progenitor cells in mammary tumorigenesis has not been fully elucidated in humans or animal models. The implications of modulating adult stem/progenitor cells in women could lead to a better understanding of not only their function, but also toward possible breast cancer prevention led us to evaluate the efficacy of rapamycin in reducing mammary stem/progenitor cell activity and malignant progression markers.MethodsWe analyzed a large number of human breast tissues for their basal and luminal cell composition with flow cytometry and their stem and progenitor cell function with sphere formation assay with respect to age and menopausal status in connection with a clinical study (NCT02642094) involving a low-dose (2 mg/day) and short-term (5–7 days) treatment of the mTOR inhibitor sirolimus. The expression of biomarkers in biopsies and surgical breast samples were measured with quantitative analysis of immunohistochemistry.ResultsSirolimus treatment significantly abrogated mammary stem cell activity, particularly in postmenopausal patients. It did not affect the frequency of luminal progenitors but decreased their self-renewal capacity. While sirolimus had no effect on basal cell population, it decreased luminal cell population, particularly in postmenopausal patients. It also significantly diminished prognostic biomarkers associated with breast cancer progression from ductal carcinoma in situ to invasive breast cancer including p16INK4A, COX-2, and Ki67, as well as markers of the senescence-associated secretary phenotype, thereby possibly functioning in preventing early breast cancer progression.ConclusionOverall, these findings indicate a link from mTOR signaling to mammary stem and progenitor cell activity and cancer progression.Trial registration This study involves a clinical trial registered under the ClinicalTrials.gov identifier NCT02642094 registered December 30, 2015.

Astrocytic phagocytosis of myelin debris and reactive characteristics in vivo and in vitro.

Persistent myelin debris can inhibit axonal regeneration, thereby hindering remyelination. Effective removal of myelin debris is essential to eliminate the interference of myelin debris in oligodendrocyte progenitor cell (OPC) activation, recruitment to demyelinating sites and/or differentiation into mature oligodendrocytes (OLs). In addition to microglia, it has been reported that astrocytic phagocytosis of myelin debris is a feature of early demyelination. In the present study, astrocytes effectively phagocytized myelin debris in vitro and in vivo. On the 5th day after injecting myelin debris into the brain, astrocytes were enriched in the area injected with myelin debris compared with microglia, and their ability to engulf myelin debris was stronger than that of microglia. When exposed to myelin debris, astrocytes phagocytizing myelin debris triggered self-apoptosis, accompanied by the activation of NF-κB, down-regulation of Nrf2, and the increase of ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (bFGF). However, the activation of astrocytic NF-κB did not influence the inflammatory cytokines IL-1β, IL-6, and TNF-α, and the anti-inflammatory factor IL-10. The proliferation of astrocytes and mobilization of OPCs in the subventricular zone were elevated on the 5th day after intracerebral injection of myelin debris. The results suggested that myelin phagocytosis of astrocytes should help improve the microenvironment and promote myelin regeneration by increasing CNTF and bFGF within the central nervous system. However, the molecular interaction of astrocytes acting as phagocytes remains to be further explored. Therefore, an improvement of astrocytes to phagocytize myelin debris may be a promising treatment measure to prevent demyelination and promote remyelination in MS and other diseases with prominent myelin injury.

CD8 T cell response and its released cytokine IFN-γ are necessary for lung alveolar epithelial repair during bacterial pneumonia.

Alveolar epithelial regeneration depends on the activity of resident quiescent progenitor cells. Alveolar epithelial type II (AT2) cells are known as the alveolar epithelial progenitor cells. They exit quiescent state, proliferate rapidly in response to injury and differentiate into alveolar epithelial type I (AT1) cells to regenerate the damaged alveolar epithelium. Although AT2 cell plasticity has been a very intense field of research, the role of CD8 T cell response and their released cytokine IFN-γ, in regulating AT2 cell plasticity and alveolar epithelial repair and regeneration after injury remains largely unknown. We used flow cytometry to quantify the amount of CD8 T cells in mouse lungs after bacterial pneumonia caused by Streptococcus pneumoniae. To determine whether CD8 T cells and their released cytokine IFN-γ are necessary for AT2 cell activity during alveolar epithelial regeneration, we performed loss of function studies using anti-CD8 or anti-IFN-γ monoclonal antibody (mAb) treatment in vivo. We assessed the effects of CD8 T cells and cytokine IFN-γ on AT2 cell differentiation capacity using the AT2- CD8 T cell co-culture system in vitro. We detected a transient wave of accumulation of CD8 T cells in mouse lungs, which coincided with the burst of AT2 cell proliferation during alveolar epithelial repair and regeneration in mice following bacterial pneumonia caused by Streptococcus pneumoniae. Depletion of CD8 T cells or neutralization of cytokine IFN-γ using anti-CD8 or anti-IFN-γ monoclonal antibody significantly reduced AT2 cell proliferation and differentiation into AT1 cells in mice after bacterial pneumonia. Furthermore, co-culture of CD8 T cells or cytokine IFN-γ with AT2 cells promoted AT2-to-AT1 cell differentiation in both murine and human systems. Conversely, blockade of IFN-γ signaling abrogated the increase in AT2-to-AT1 cell differentiation in the AT2- CD8 T cell co-culture system. Our data demonstrate that CD8 T-cell response and cytokine IFN-γ are necessary for promoting AT2 cell activity during alveolar epithelial repair and regeneration after acute lung injury caused by bacterial pneumonia.

THU043 Single-cell Transcriptomics Show Sex-specific Responses To A High Fat Diet, Under Thermoneutral Conditions, Pointing To Reduced Pituitary Plasticity

Abstract Disclosure: T.K. Miles: None. A.N. Lagasse: None. A.K. Odle: None. S.S. Lira: None. M.M. Syed: None. N. Akhter: None. M.C. MacNicol: None. A.M. MacNicol: None. G.V. Childs: None. Hyperinsulinemia caused by a high fat diet (HFD) directly suppressed pituitary growth hormone (Gh) and growth hormone releasing hormone receptor (Ghrhr) mRNAs (Luque and Kineman, 2006). To study the broader impact of a HFD, we exposed 3 groups of FVB.129P mice to 60% HFD for 10 or 15 wks at thermoneutrality (TN) (30 C), or 15 wks at room temperature (RT). Pituitary transcript expression was analyzed with single-cell RNA-seq. HFD-fed mice gained >20 g by 12 wks, showing increased serum leptin (1.8-2X), insulin (2-8X) and IL-6 (4-6X), decreased glucose clearance after a glucose challenge and decreased respiratory quotient indicating fat burning. Activity was unaffected by HFD. Rank plot analyses showed reduced amplitude GH pulses in HFD females fed at RT (15 wks), but not in mice fed at TN. Serum levels of other pituitary hormones were unaffected in HFD females. In HFD males, only serum TSH was elevated significantly (1.8X, 15 wks, TN). Analysis of the scRNA-seq data detected separate clusters representing each cell type. Two multihormonal cell clusters evident in control females mostly disappeared following HFD (15 wks, RT). At the threshold cutoff of avg Log2FC±0.5 with adj p<0.05, female somatotrope clusters showed reduced Pomc, Cga, and ribosomal proteins. Female lactotrope clusters exhibited decreased Gh mRNA (avg Log2FC -1.8 or -2.5) and galanin at 10 wks (TN). At 15 wks (TN), lactotrope clusters showed significant changes in 46 transcripts including reductions in Prl, 29 ribosomal proteins, galanin, and 3 transcriptional regulators. Notably, this cluster showed an increase in synaptotagmin-4 (Syt4), which is also elevated in oxytocin neurons following a HFD. Stem cell clusters in females were strongly impacted by HFD after 15 weeks (TN) with 94 reduced transcripts of which 48 were involved in cytoplasmic translation and ribosome biogenesis and >20 were involved with signaling and cell differentiation. In males, whereas changes in most clusters fell below the threshold cutoffs, stem cell clusters had 13 reduced transcripts after 10 or 15 weeks, most of which involved immune system processes including defense responses and antigen processing and presentation. The GH deficiency in mice fed under thermoneutral conditions was not as severe as that reported by Luque and Kineman, perhaps due to differences in strain, sex, and/or the environmental temperature. The reduced expression of multihormonal clusters, Gh by lactotropes and corticotropes, and Cga and Pomc by somatotropes suggests that HFD may reduce plasticity and activity of progenitor cells in the pituitary. The changes in lactotropes and somatotropes also indicate that HFD may compromise ribosome function and cytoplasmic translation. The reduced transcriptome in stem cells in both males and females suggests HFD interference with cell differentiation and pituitary plasticity as well as dysfunctional immune defense responses. Presentation: Thursday, June 15, 2023

Human iPSC-derived endothelial cells promote CNS remyelination via BDNF and mTORC1 pathway.

Damage of myelin is a component of many diseases in the central nervous system (CNS). The activation and maturation of the quiescent oligodendrocyte progenitor cells (OPCs) are the crucial cellular processes for CNS remyelination, which is influenced by neuroinflammation in the lesion microenvironment. Endothelial cells derived from human induced pluripotent stem cells (hiPSC-ECs) have shown promise in restoring function in various preclinical animal models. Here we ask whether and whether transplantation of hiPSC-ECs could benefit remyelination in a mouse model of CNS demyelination. Our results show that in vitro, hiPSC-ECs increase OPC proliferation, migration and differentiation via secreted soluble factors including brain-derived neurotrophic factor (BDNF). hiPSC-ECs also promote the survival of oligodendrocyte lineage cells in vitro and in vivo. Transplantation of hiPSC-ECs into a toxin-induced demyelination lesion in mouse corpus callosum (CC) leads to increased density of oligodendrocyte lineage cells and level of myelin in demyelinated area, correlated with a decreased neuroinflammation and an increased proportion of pro-regenerative M2 phenotype in microglia/macrophages. The hiPSC-EC-exposed oligodendrocyte lineage cells showed significant increase in the level of phosphorylated S6 ribosomal protein (pS6) both in vitro and in vivo, indicating an involvement of mTORC1 pathway. These results suggest that hiPSC-ECs may benefit myelin protection and regeneration which providing a potential source of cell therapy for a wide range of diseases and injuries associated with myelin damage.

S1PR-1/5 modulator RP-101074 shows beneficial effects in a model of central nervous system degeneration.

In multiple sclerosis (MS), chronic disability primarily stems from axonal and neuronal degeneration, a condition resistant to conventional immunosuppressive or immunomodulatory treatments. Recent research has indicated that selective sphingosine-1-phosphate receptor S1PR-1 and -5 modulators yield positive effects in progressive MS and mechanistic models of inflammation-driven neurodegeneration and demyelination. In this study, the S1PR-1/-5 modulator RP-101074 was evaluated as a surrogate for ozanimod in the non-inflammatory, primary degenerative animal model of light-induced photoreceptor loss (LI-PRL) in CX3CR1-GFP mice to assess potential neuroprotective effects, independent of its immunomodulatory mechanism of action. Prophylactic administration of RP-101074 demonstrated protective effects in the preclinical, non-inflammatory LI-PRL animal model, following a bell-shaped dose-response curve. RP-101074 treatment also revealed activity-modulating effects on myeloid cells, specifically, CX3CR1+ cells, significantly reducing the marked infiltration occurring one week post-irradiation. Treatment with RP-101074 produced beneficial outcomes on both retinal layer thickness and visual function as evidenced by optical coherence tomography (OCT) and optomotor response (OMR) measurements, respectively. Additionally, the myelination status and the quantity of neural stem cells in the optic nerve suggest that RP-101074 may play a role in the activation and/or recruitment of neural stem cells and oligodendrocyte progenitor cells, respectively. The data from our study suggest that RP-101074 may have a broader role in MS treatment beyond immunomodulation, potentially offering a novel approach to mitigate neurodegeneration, a core contributor to chronic disability in MS.

Abstract P1094: Mesenchymal Stem Cells Educate Lung Megakaryocytes To Favor Cardiac Repair Post Myocardial Infarction

Stem cell therapy in myocardial infarction (MI) have mixed clinical results, and the mechanism of its potential benefits is uncertain. Intravenously administrated mesenchymal stem cells (MSCs) are trapped in the lung capillary bed and hardly reach to the heart. Despite their extremely low retention in the heart, cardiac repair benefits are still seen in multiple clinical trials. There is a large knowledge gap exists regarding the interconnection and bidirectional pathways between heart and lung. It has been established that lung is a reservoir of megakaryocytes (MKs), which produce platelets. We found that MSCs trapped in the lung capillary altered the transcription profiles of lung MKs and circulating platelets. Single cell analysis revealed that MSCs reduced the activation of megakaryocyte progenitor cells (MKp) and promoted MK maturation in the lung. Additionally, MSC treatment reduced the activation of immune cells and the ratio of proinflammatory monocytes in the lung and macrophage infiltration in the infarcted area. CellChat analysis demonstrated that reduced communication between lymphocytes and MKp after MSC treatment. Lastly, we demonstrated that cell-cell contact was needed for this education process, and MSC secretome didn’t promote the megakaryopoiesis process in vitro and in vivo . Intravenously injection of MSCs showed a better therapeutic effect compared to MSC secretome in MI mice. Collectively, this study unevils the role of pulmonary MK in lung-heart crosstalk and the interaction of MSCs trapped in the lung tissue with pulmonary MK during MI. Scheme 1. Schematic illustration of heart-lung crosstalk and the role of lung MK during MI.

SAT-369 - Hepatic progenitor cell activation through the interaction of mesenchymal stem cells and liver sinusoidal endothelial cells

SAT-369 - Hepatic progenitor cell activation through the interaction of mesenchymal stem cells and liver sinusoidal endothelial cells

Modified Risk Tobacco Products and Cardiovascular Repair: Still Very "Smoky".

Smoking habits represent a cardiovascular risk factor with a tremendous impact on health. Other than damaging differentiated and functional cells of the cardiovascular system, they also negatively affect reparative mechanisms, such as those involved in cardiac fibrosis and in endothelial progenitor cell (EPC) activation. In recent years, alternative smoking devices, dubbed modified tobacco risk products (MRPs), have been introduced, but their precise impact on human health is still under evaluation. Also, they have not been characterized yet about the possible negative effects on cardiovascular reparative and regenerative cells, such as EPCs or pluripotent stem cells. In this perspective, we critically review the still scarce available data on the effects of MRPs on molecular and cellular mechanisms of cardiovascular repair and regeneration.

Elevated Serum Alpha-fetoprotein Levels in Non-alcoholic Steatohepatitis: Possible Molecular Mechanisms and Potential Clinical Significance

With the improvement of living standards in recent years, up to 90% of obese patients have nonalcoholic fatty liver disease (NAFLD). The number of nonalcoholic steatohepatitis (NASH)-related deaths will gradually increase, and NASH is expected to be the most common cause of liver-related deaths in the future. Therefore, there is an urgent need to find effective and reliable serum biomarkers to distinguish simple hepatic steatosis (SS) from NASH. Liver cell regeneration, oxidative stress-induced DNA methylation, and biliary epithelial cell proliferation were reported to increase serum alpha-fetoprotein (AFP) levels. AFP has long been used as a tool to monitor liver cancer. However, the function of AFP in NAFLD, especially NASH, has not been clarified. Moreover, whether an elevated AFP level indicates the occurrence of NASH or serves as a serum biomarker remains to be elucidated. The miRNA-122 pathway, DNA methylation and DNA damage, and activation of resident stem cells and/or progenitor cells in the liver, as well as necrosis, regeneration, and repair of liver cells, may contribute to slight increases in AFP levels in the development of NASH in patients with NAFLD. Furthermore, mildly elevated AFP levels may indicate the development of NASH. This review explores the role of elevated AFP levels in the development of NASH, with a specific focus on the underlying molecular mechanisms and the clinical significance.

Abstract 3496: p53 mutation biases SCJ progenitor cells towards dysplasia rather than metaplasia in Barrett's esophagus

Abstract Objectives: Barrett's esophagus (BE) is a form of metaplasia that involves the aberrant differentiation of stem or progenitor cells at the squamocolumnar junction (SCJ). p53 mutation occurs early in BE cases that do progress to dysplasia or esophageal adenocarcinoma. Here, we aimed to elucidate the mechanisms by which p53 mutation promotes the early neoplastic progression of cardia progenitor cells in a mouse model of BE (L2-IL1β). Methods: We generated the transgenic mice L2-IL1β; Cck2r-CreERT2; tdTomato; p53R172H/+ to determine the effect of p53 mutation on Cck2r+ cardia progenitor cell activity. Lineage tracing and pathological assessment were performed in combination at different time points to explore the effects of p53 mutation on development of precancerous lesions in the cardia glands. Organoids were derived from sorted Cck2r+ cardia cells with or without p53 mutation. Organoid forming capacity and growth assessment were tested to assess stemmness potential. Organoids were treated with the DNA damaging agent N-methyl-N-nitrosourea (MNU) to test the resistance of p53 mutant organoids to carcinogenic injury. Orthotopic transplantation of organoids into immunodeficient (NSG) mice was performed to study the influence of p53 mutation on growth in vivo. Single-cell RNA sequencing (scRNA-seq) analysis of precancerous lesions and bulk RNA-seq analysis of cardia organoids from BE mice were carried out to investigate the effects of p53 mutation on key regulatory pathways. Results: Characterization of pathological progression combined with lineage tracing showed that over time, most p53 mutant cells did not undergo metaplasia but progressed to dysplasia in the cardia glands of IL1β mice. This suggests that the dysplastic cells likely arose directly from stem or progenitor cells rather than progressing first through metaplasia, a finding consistent with the scRNA-seq analysis. In vitro, p53 mutation increased the number and size of organoids derived from Cck2r+ progenitor cells from IL1β mice, suggesting an expansion of stem or progenitor cells. Furthermore, p53 mutation led to increased resistance of these organoids to MNU. Orthotopic transplantation experiments demonstrated that p53 mutation increased survival and proliferation of organoids in the stroma of immunodeficient murine stomach. Moreover, p53 mutant transplanted organoids retained dysplastic features with little to no metaplastic events relative to their WT counterparts. Computational analyses suggested that these heterotypic gains function in vivo and in vitro are relate to their inhibitory effect of stem cell differentiation through Notch signaling activation. Conclusion: These findings suggest that p53R172H/+ mutation in SCJ progenitor cells directly promotes their progression toward dysplasia, rather than differentiating into metaplasia, due in part through activation of Notch signaling to maintain stemness. Citation Format: Guodong Lian, Ermanno Malagola, Junfei Zhao, Richard A. Friedman, Leah B. Zamechek, Timothy C. Wang. p53 mutation biases SCJ progenitor cells towards dysplasia rather than metaplasia in Barrett's esophagus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3496.

Low-dose curcumin enhances hippocampal neurogenesis and memory retention in young mice.

Adult neurogenesis generates new functional neurons from adult neural stem cells in various regions, including the subventricular zone (SVZ) of the lateral ventricles and subgranular zone (SGZ) of hippocampal dentate gyrus (DG). Available evidence shows hippocampal neurogenesis can be negatively or positively regulated by dietary components. In a previous study, we reported that curcumin (diferuloylmethane; a polyphenolic found in curry spice) stimulates the proliferation of embryonic neural stem cells (NSCs) by activating adaptive cellular stress responses. Here, we investigated whether subchronic administration of curcumin (once daily at 0.4, 2, or 10mg/kg for 14 days) promotes hippocampal neurogenesis and neurocognitive function in young (5-week-old) mice. Oral administration of low-dose curcumin (0.4mg/kg) increased the proliferation and survival of newly generated cells in hippocampus, but surprisingly, high-dose curcumin (10mg/kg) did not effectively upregulate the proliferation or survival of newborn cells. Furthermore, hippocampal BDNF levels and phosphorylated CREB activity were elevated in only low-dose curcumin-treated mice. Passive avoidance testing revealed that low-dose curcumin increased cross-over latency times, indicating enhanced memory retention, and an in vitro study showed that low-concentration curcumin increased the proliferative activity of neural progenitor cells (NPCs) by upregulating NF1X levels. Collectively, our findings suggest that low-dose curcumin has neurogenic effects and that it may prevent age and neurodegenerative disease-related cognitive deficits.

Osteoregenerative Potential of 3D-Printed Poly ε-Caprolactone Tissue Scaffolds In Vitro Using Minimally Manipulative Expansion of Primary Human Bone Marrow Stem Cells.

The repair of orthopedic and maxillofacial defects in modern medicine currently relies heavily on the use of autograft, allograft, void fillers, or other structural material composites. This study examines the in vitro osteo regenerative potential of polycaprolactone (PCL) tissue scaffolding, fabricated via a three-dimensional (3D) additive manufacturing technology, i.e., a pneumatic micro extrusion (PME) process. The objectives of this study were: (i) To examine the innate osteoinductive and osteoconductive potential of 3D-printed PCL tissue scaffolding and (ii) To perform a direct in vitro comparison of 3D-printed PCL scaffolding with allograft Allowash® cancellous bone cubes with regards to cell-scaffold interactions and biocompatibility with three primary human bone marrow (hBM) stem cell lines. This study specifically examined cell survival, cell integration, intra-scaffold cell proliferation, and differentiation of progenitor cells to investigate the potential of 3D-printed PCL scaffolds as an alternative to allograft bone material for the repair of orthopedic injuries. We found that mechanically robust PCL bone scaffolds can be fabricated via the PME process and the resulting material did not elicit detectable cytotoxicity. When the widely used osteogenic model SAOS-2 was cultured in PCL extract medium, no detectable effect was observed on cell viability or proliferation with multiple test groups showing viability ranges of 92.2% to 100% relative to a control group with a standard deviation of ±10%. In addition, we found that the honeycomb infill pattern of the 3D-printed PCL scaffold allowed for superior mesenchymal stem-cell integration, proliferation, and biomass increase. When healthy and active primary hBM cell lines, having documented in vitro growth rates with doubling times of 23.9, 24.67, and 30.94 h, were cultured directly into 3D-printed PCL scaffolds, impressive biomass increase values were observed. It was found that the PCL scaffolding material allowed for biomass increase values of 17.17%, 17.14%, and 18.18%, compared to values of 4.29% for allograph material cultured under identical parameters. It was also found that the honeycomb scaffold infill pattern was superior to the cubic and rectangular matrix structures, and provided a superior microenvironment for osteogenic and hematopoietic progenitor cell activity and auto-differentiation of primary hBM stem cells. Histological and immunohistochemical studies performed in this work confirmed the regenerative potential of PCL matrices in the orthopedic setting by displaying the integration, self-organization, and auto-differentiation of hBM progenitor cells within the matrix. Differentiation products including mineralization, self-organizing "proto-osteon" structures, and in vitro erythropoiesis were observed in conjunction with the documented expression of expected bone marrow differentiative markers including CD-99 (>70%), CD-71 (>60%), and CD-61 (>5%). All of the studies were conducted without the addition of any exogenous chemical or hormonal stimulation and exclusively utilized the abiotic and inert material polycaprolactone; setting this work apart from the vast majority of contemporary investigations into synthetic bone scaffold fabrication In summary, this study demonstrates the unique clinical potential of 3D-printed PCL scaffolds for stem cell expansion and incorporation into advanced microstructures created via PME manufacturing to generate a physiologically inert temporary bony defect graft with significant autograft features for enhanced end-stage healing.

Rngtt governs biliary-derived liver regeneration initiation by transcriptional regulation of mTORC1 and Dnmt1 in zebrafish.

In cases of end-stage liver diseases, the proliferation of existing hepatocytes is compromised, a feature of human chronic liver disease, in which most hepatocytes are dysfunctional. So far, liver transplantation represents the only curative therapeutic solution for advanced liver diseases, and the shortage of donor organs leads to high morbidity and mortality worldwide. The promising treatment is to prompt the biliary epithelial cells (BECs) transdifferentiation. However, the critical factors governing the initiation of BEC-derived liver regeneration are largely unknown. The zebrafish has advantages in large-scale genetic screens to identify the critical factors involved in liver regeneration. Here, we combined N-ethyl-N-nitrosourea screen, positional cloning, transgenic lines, antibody staining, and in situ hybridization methods and identified a liver regeneration defect mutant ( lrd ) using the zebrafish extensive liver injury model. Through positional cloning and genomic sequencing, we mapped the mutation site to rngtt . Loss of rngtt leads to the defects of BEC dedifferentiation, bipotential progenitor cell activation, and cell proliferation in the initiation stage of liver regeneration. The transdifferentiation from BECs to hepatocytes did not occur even at the late stage of liver regeneration. Mechanically, Rngtt transcriptionally regulates the attachment of mRNA cap to mTOR complex 1 (mTORC1) components and dnmt1 to maintain the activation of mTORC1 and DNA methylation in BECs after severe liver injury and prompt BEC to hepatocyte conversion. Furthermore, rptor and dnmt1 mutants displayed the same liver regeneration defects as rngtt mutation. In conclusion, our results suggest Rngtt is a new factor that initiates BEC-derived liver regeneration.

Функціональна активність гемопоетичних клітин-попередників кордової крові у довготривалій культурі на гідрогелевій підложці in vitro

Development of new and improvement of existing models of long-term cultivation of hematopoietic stem cells (HSC) and cord blood progenitor cells is one of the most important directions in modern cell biology, related to obtaining a sufficient amount of hematopoietic tissue for experimental and clinical use. The aim of the work was to investigate the functional activity of hematopoietic progenitor cells from cord blood during long-term cultivation on a soft polyacrylamide gel substrate in the presence of a cytokine complex and to determine the advantages of this method of maintaining hematopoiesis for further use in transplantation. To realize this purpose, the following methods were used: the method of long-term cultivation in vitro on a hydrogel substrate, the method of colony formation in semi-liquid agar, cytological research methods, light and inverted microscopy, statistical research methods. Due to the use of the hydrogel substrate together with the cytokine complex, a high proliferative activity of hematopoietic cells is observed, which is reflected in their expansion during long-term cultivation (5 weeks), as well as of high colony-forming activity (521.5 ± 7.5.105 per explanted cells). Thus, the stiffness of the substrate must be taken into account for the expansion of stem cells and their immediate descendants. The presence of a soft substrate made of polyacrylamide gel along with a complex of cytokines ensures the expansion of hematopoietic cells due to the long- term support of hematopoiesis.