Progenitor Cell Niche Research Articles

Jamming of nephron-forming niches in the developing mouse kidney creates cyclical mechanical stresses.

Urinary collecting tubules form during kidney embryogenesis through the branching of the ureteric bud epithelium. A travelling mesenchyme niche of nephron progenitor cells caps each branching ureteric bud tip. These 'tip domain' niches pack more closely over developmental time and their number relates to nephron endowment at birth. Yet, how the crowded tissue environment impacts niche number and cell decision-making remains unclear. Here, through experiments and mathematical modelling, we show that niche packing conforms to physical limitations imposed by kidney curvature. We relate packing geometries to rigidity theory to predict a stiffening transition starting at embryonic day 15 in the mouse, validated by micromechanical analysis. Using a method to estimate tip domain 'ages' relative to their most recent branch events, we find that new niches overcome mechanical resistance as they branch and displace neighbours. This creates rhythmic mechanical stress in the niche. These findings expand our understanding of kidney development and inform engineering strategies for synthetic regenerative tissues.

β-catenin Signaling Drives Intestinal Epithelial Production of Interleukin-33

Background: Interleukin (IL)-33 is a key responder to intestinal infection, injury, and inflammation. It drives protective epithelial re-modeling via a stromal-epithelial circuit. However, we know little about the regulation of epithelial IL-33 at homeostasis and in disease. Our group previously found that colonic epithelial production of IL-33 is restrained by the inflammation-responsive signaling regulator, Sprouty2. Loss of Sprouty2 activates GSK3β (a key mediator of canonical Wnt/β-catenin signaling) and induces IL-33 in the colon, but the specific role of β-catenin and Wnt pathway components involved in this induction are unknown. Hypothesis: Canonical Wnt/β-catenin signaling induces colonic epithelial expression of IL-33. Methods: Bulk and single-cell RNAseq were performed on colons from mice with an intestinal epithelial-specific Sprouty2 deletion (Spry2IEKO) or littermate controls and subjected to gene enrichment and Panther pathway analyses. Colonic epithelial cell cultures were treated with Wnt3a and R-spondin to activate β-catenin signaling; small molecule inhibitors were used to inhibit CBP- or p300-mediated β-catenin signaling arms. IL-33 levels were assayed by qPCR and ELISA. Results: Analysis of significantly altered genes in Spry2IEKO colons by Panther analysis found an enrichment in genes regulated by canonical Wnt signaling (FDR 3.72E-02), correlating with increased IL-33 observed in these mice. Analysis of single-cell RNAseq suggests colonic epithelial IL-33 is predominantly expressed in the stem and progenitor cell niche. Colonic epithelial cells treated with Wnt ligand showed a significant induction in IL-33 (p<0.05) at both RNA and protein levels. Inhibition of CBP-mediated but not p300-mediated β-catenin signaling prevented IL-33 induction, correlating with decreased expression of the CBP inhibitor, Psen1, in Spry2IEKO colons. Conclusion: CBP-mediated β-catenin signaling promotes colonic epithelial expression of IL-33. Acute inflammation-mediated loss of Sprouty2 drives IL-33 induction in the stem cell niche as a protective response to injury or infection. NIH K01DK131390 (MS); Crohn's and Colitis Foundation CDA (MS); NIH R01DK135954 (CL); NIH R01DK119694 (MF). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hepatic stellate cells and their role in the formation of the progenitor cell niche

The processes of proliferation and differentiation of progenitor and stem cells in the body are ensured by a specific microenvironment, the stem cell niche. Universal components have been identified for all niches: supporting cells, extracellular matrix, and soluble biological factors. A niche is a dynamic system whose activity depends on regeneration needs.The review presents data on the structure of the hepatic stem cell niche and one of its main components – stellate cells and their role in pathology.

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.

In Vivo Reprogramming of Adult Liver Sinusoidal Vascular Endothelial Cells into a Hematopoietic Stem and Progenitor Cell Niche

Hematopoietic stem and progenitor cells (HSPCs) reside in specialized microenvironments known as the HSPC niche. Vascular endothelial cells (ECs) are one of the most abundant and significant components of the niche, providing known supportive factors such as E-selectin, CXCL12, and SCF. During marrow stress, extramedullary hematopoiesis can occur in the liver or spleen, but the process is inefficient. The sinusoidal vessels of the liver share many similarities with marrow sinusoids. Here, we aim to identify the transcription factor (TF) code that specifies the vascular EC fate in HSPC marrow niche by conducting differential gene expression analysis with the non-niche supportive ECs of the liver. We performed scRNAseq analysis on FACS-isolated ECs from adult zebrafish kidney marrow and liver. We found that these ECs express conserved sinusoidal genes, such as sele, flt4 and gpr182. However, marrow ECs have significantly higher expression of HSPC niche supportive genes, including mrc1a, lyve1b and cxcl12a. We further identified putative TFs that are uniquely upregulated in the marrow ECs, namely tfec, mafbb, foxp4, irf8 and hoxb8a. The expression of these candidate TFs was conserved in the mammalian bone marrow ECs, as shown by relevant mouse bone marrow scRNA-seq and human bone marrow RNA-seq analyses. To determine whether these candidate TFs can functionally specify a HSPC niche EC fate, we selectively overexpressed them using a zebrafish liver sinusoidal EC-specific enhancer in vivo. Upon the overexpression of tfec and mafbb together, we found adult zebrafish liver sinusoidal ECs significantly upregulated key genes known for HSPC niche supportive functions in both embryonic and adult zebrafish HSPC niches, including mrc1a (log2FC=4.9, p<0.005) and lyve1b (log2FC=3.6, p<0.005) and dab2 (log2FC=5.0, p<0.005). Overexpression of TFEC and MAFB in human iPSC-derived ECs showed upregulation of HPSC niche supportive genes that we previously identified from zebrafish in vivo studies, including LYVE1 (log2FC=5.6, p<0.005) and DAB2 (log2FC=1.1, p<0.005) . Notably, previously published HSPC niche genes, including CXCL12(log2FC=5.1 p< 0.005) , PTN (log2FC=2.6 p<0.005) and JAG1 (log2FC=1.7 p<0.005), are also upregulated upon TFEC and MAFB overexpression. Furthermore, gene ontology analysis revealed an enrichment of genes related to cell adhesion (p<0.005), migration (p<0.005), and extravasation pathways (p=0.014). A colony-forming unit (CFU) assay of CD34+CD45+ HSPCs co-cultured with TFEC and MAFB overexpressing hiPSC-derived ECs had significantly greater numbers of CFUs compared to those co-cultured with hiPSC-derived ECs without the TF overexpression: CFU-GEMM (23.3±2.4 vs. 10.0±2.1, p=0.004), CFU-G (89.3±13.4 vs. 46.0±4.2, p=0.027), and CFU-E (42.3±3.3 vs. 26.7±0.9, p=0.021). Our findings revealed a TF code that specifies the HSPC niche supportive fate of vascular ECs and distinguishes vascular ECs in the hematopoietic niche vs. non-niche organs. These results may translate into novel therapies targeting the sinusoidal vascular niches to better stimulate and support human HSPCs.

Feeling at home: Identifying a human thymic epithelial progenitor cell niche

T cell development relies on a supportive epithelial microenvironment. Embryonic and postnatal epithelial progenitors have been identified in mice, but not humans. In this issue of Developmental Cell, Raggazzini etal. use scRNAseq, spatial sequencing, and clonogenic assays to identify a putative bipotent TEPC in pediatric human thymic tissue.

Abstract A011: Single-cell RNA sequencing reveals a potential renal tumor progenitor in conditional Vhlh knockout mouse model and ccRCC clinical samples

Abstract Introduction: The study aims to identify the progenitor of clear-cell renal cell carcinoma (ccRCC). Background: Inactivation of tumor suppressor gene VHL is the driver for the pathogenesis of ccRCC, the majority subtype of kidney tumors. However, it has also been observed that not all VHL-deficient cells develop into tumors. It is therefore likely that additional characteristics are required for ccRCC formation. Accumulating evidence suggests that tumor formation is initiated by a small pool of cells with tumor-initiating capacity, which are termed tumor progenitor cells (TPCs). Yet, the concept of TPCs in ccRCC has not been fully validated. Methods: LSL-tdTomato reporter was used to trace Vhlh mutant cells in the prior pre-malignant ccRCC mouse model, in which Vhlh was conditionally knocked out (VhlhKO) in renal epithelial cells under the control of Hoxb7-Cre. We also utilized single-cell RNA sequencing (scRNA-seq) to investigate the diversity of Vhlh-deficient renal tubule cells. Results: We show that the tubule phenotypes (traced by the tdTomato reporter) are associated with Vhlh inactivation; the abnormal tdTomato+ cells in Vhlh KO mice exhibit clear-cell phenotype, hyperplasia and multi-layered epithelial cell clusters. scRNA-seq was then used to analyze isolated tdTomato+ cells from Vhlh KO and wild-type (WT) kidneys. The results reveal that inactivation of Vhlh generates an expanded population of principal collecting duct cells characterized by the expression of keratin 19 (Krt19). These Krt19+ cells highly express stem cell-related genes (Wnt4, Sox4, Klf4, Ly6a). They also express multiple tubule segment-specific markers, suggesting the process of dedifferentiation. The expression of Krt19 is strikingly increased and colocalized with a subpopulation of tdTomato+ cells in Vhlh KO kidney, compared with WT where Krt19+ cells are a rare population scattered in tubule epithelia. Colocalization of KRT19 and CAIX in ccRCC samples confirms the ccRCC identity of these KRT19+ cells. High expression of KRT19 is significantly associated with poor overall survival of ccRCC patients. On kidney tissue microarray, KRT19 is strongly expressed in the early-stage peri-tumor tissue with the presence of immune cell infiltration and lipid droplets in the vicinity. In the high-grade ccRCC, KRT19 is expressed in the typical ccRCC cells and in multi-layered adenomas. Notably, Krt19+ progenitor-like cells are found in the fibrotic region with immune cell infiltration in Vhlh mutant kidney. The direct contact between macrophages and Krt19+ cells in mutant kidney, but not in WT, implies that macrophages may be a component in the progenitor-cell niche when Vhlh is conditionally knocked out. Conclusion: We suspect that VHL loss-of-function in the rare tissue progenitor cells may generate a tumor progenitor subset. Elucidation of the role of KRT19 in the context of VHL inactivation and characterization of the stem/progenitor cell niche are of importance as they will shed new lights on early diagnosis and therapeutic intervention. Citation Format: Dinh-Xuan Pham, Chen-Yun Chen, Hieu-Huy Nguyen-Tran, Tien Hsu. Single-cell RNA sequencing reveals a potential renal tumor progenitor in conditional Vhlh knockout mouse model and ccRCC clinical samples [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A011.

The tumor suppressor CREBBP and the oncogene MYCN cooperate to induce malignant brain tumors in mice

The tumor suppressor and chromatin modifier cAMP response element-binding protein binding protein (CREBBP) and v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), a member of the MYC oncogene family, are critically involved in brain development. Both genes are frequently mutated in the same tumor entities, including high-grade glioma and medulloblastoma. Therefore, we hypothesized that alterations in both genes cooperate to induce brain tumor formation. For further investigation, hGFAP-cre::CrebbpFl/Fl::lsl-MYCN mice were generated, which combine Crebbp deletion with overexpression of MYCN in neural stem cells (NSCs). Within eight months, these animals developed aggressive forebrain tumors. The first tumors were detectable in the olfactory bulbs of seven-day-old mice. This location raises the possibility that presumptive founder cells are derived from the ventricular-subventricular zone (V-SVZ). To examine the cellular biology of these tumors, single-cell RNA sequencing was performed, which revealed high intratumoral heterogeneity. Data comparison with reference CNS cell types indicated the highest similarity of tumor cells with transit-amplifying NSCs or activated NSCs of the V-SVZ. Consequently, we analyzed V-SVZ NSCs of our mouse model aiming to confirm that the tumors originate from this stem cell niche. Mutant V-SVZ NSCs showed significantly increased cell viability and proliferation as well as reduced glial and neural differentiation in vitro compared to control cells. In summary, we demonstrate the oncogenic potential of a combined loss of function of CREBBP and overexpression of MYCN in this cell population. hGFAP-cre::CrebbpFl/Fl::lsl-MYCN mice thus provide a valuable tool to study tumor-driving mechanisms in a key neural stem/ progenitor cell niche.

PDGF receptor signal mediates the contribution of Nestin-positive cell lineage to subcutaneous fat development

The beiging of white adipose tissue (WAT) is expected to improve systemic metabolic conditions; however, the regulation and developmental origin of this process remain insufficiently understood. In the present study, the implication of platelet-derived growth factor receptor alpha (PDGFRα) was examined in the beiging of inguinal WAT (ingWAT) of neonatal mice. Using in vivo Nestin expressing cell (Nestin+) lineage tracing and deletion mouse models, we found that, in the mice with Pdgfra gene inactivation in Nestin+ lineage (N-PRα-KO mice), the growth of inguinal WAT (ingWAT) was suppressed during neonatal periods as compared with control wild-type mice. In the ingWAT of N-PRα-KO mice, the beige adipocytes appeared earlier that were accompanied by the increased expressions of both adipogenic and beiging markers compared to control wild-type mice. In the perivascular adipocyte progenitor cell (APC) niche of ingWAT, many PDGFRα+ cells of Nestin+ lineage were recruited in Pdgfra-preserving control mice, but were largely decreased in N-PRα-KO mice. This PDGFRα+ cell depletion was replenished by PDGFRα+ cells of non-Nestin+ lineage, unexpectedly resulting in an increase of total PDGFRα+ cell number in APC niche of N-PRα-KO mice over that of control mice. These represented a potent homeostatic control of PDGFRα+ cells between Nestin+ and non-Nestin+ lineages that was accompanied by the active adipogenesis and beiging as well as small WAT depot. This highly plastic nature of PDGFRα+ cells in APC niche may contribute to the WAT remodeling for the therapeutic purpose against metabolic diseases.

Bone Marrow Adipose Tissue: Regulation of Osteoblastic Niche, Hematopoiesis and Hematological Malignancies.

Bone marrow adipose tissue (BMAT) creates a specific microniche within multifunctional bone marrow (BM) ecosystem which imposes changes in surrounding cells and at systemic level. Moreover, BMAT contributes to spatial and temporal separation and metabolic compartmentalization of BM, thus regulating BM homeostasis and diseases. Recent findings have identified novel progenitor subsets of bone marrow adipocytes (BMAd)s recruited during the BM adipogenesis within different skeletal and hematopoietic stem cell niches. Potential of certain mesenchymal BM cells to differentiate into both osteogenic and adipogenic lineages, contributes to the complex interplay of BMAT with endosteal (osteoblastic) niche compartments as an important cellular player in bone tissue homeostasis. Targeting and ablation of BMAT cells at certain states might be an optional and promising strategy for improvement of bone health. Additionally, recent findings demonstrated spatial distribution of BMAds related to hematopoietic cells and pointed out important functional roles in the vital processes such as long-term hematopoiesis. BM adipogenesis appears to be an emergency phenomenon that follows the production of hematopoietic stem and progenitor cell niche factors, thus regulating physiological, stressed, and malignant hematopoiesis. Lipolytic and secretory activity of BMAds can influence survival and proliferation of hematopoietic cells at different maturation stages. Due to their different lipid status, constitutive and regulated BMAds are important determinants of normal and malignant hematopoietic cells. Further elucidation of cellular and molecular players involved in BMAT expansion and crosstalk with malignant cells is of paramount importance for conceiving the new therapies for improvement of BM health.

Alcam-a and Pdgfr-α are essential for the development of sclerotome-derived stromal cells that support hematopoiesis

Mesenchymal stromal cells are essential components of hematopoietic stem and progenitor cell (HSPC) niches, regulating HSPC proliferation and fates. Their developmental origins are largely unknown. In zebrafish, we previously found that the stromal cells of the caudal hematopoietic tissue (CHT), a niche functionally homologous to the mammalian fetal liver, arise from the ventral part of caudal somites. We have now found that this ventral domain is the sclerotome, and that two markers of mammalian mesenchymal stem/stromal cells, Alcam and Pdgfr-α, are distinctively expressed there and instrumental for the emergence and migration of stromal cell progenitors, which in turn conditions the proper assembly of the vascular component of the CHT niche. Furthermore, we find that trunk somites are similarly dependent on Alcam and Pdgfr-α to produce mesenchymal cells that foster HSPC emergence from the aorta. Thus the sclerotome contributes essential stromal cells for each of the key steps of developmental hematopoiesis.

Artificial Intelligence–Derived Electrocardiogram Assessment of Cardiac Age and Molecular Markers of Senescence in Heart Failure

ObjectiveTo ascertain whether heart failure (HF) itself is a senescent phenomenon independent of age, and how this is reflected at a molecular level in the circulating progenitor cell niche, and at a substrate level using a novel electrocardiogram (ECG)–based artificial intelligence platform. Patients and MethodsBetween October 14, 2016, and October 29, 2020, CD34+ progenitor cells were analyzed by flow cytometry and isolated by magnetic-activated cell sorting from patients of similar age with New York Heart Association functional classes IV (n = 17) and I-II (n = 10) heart failure with reduced ejection fraction and healthy controls (n = 10). CD34+ cellular senescence was quantitated by human telomerase reverse transcriptase expression and telomerase expression by quantitative polymerase chain reaction, and senescence-associated secretory phenotype (SASP) protein expression assayed in plasma. An ECG-based artificial intelligence (AI) algorithm was used to determine cardiac age and difference from chronological age (AI ECG age gap). ResultsCD34+ counts and telomerase expression were significantly reduced and AI ECG age gap and SASP expression increased in all HF groups compared with healthy controls. Expression of SASP protein was closely associated with telomerase activity and severity of HF phenotype and inflammation. Telomerase activity was more closely associated with CD34+ cell counts and AI ECG age gap. ConclusionWe conclude from this pilot study that HF may promote a senescent phenotype independent of chronological age. We show for the first time that the AI ECG in HF shows a phenotype of cardiac aging beyond chronological age, and appears to be associated with cellular and molecular evidence of senescence.

A single-cell transcriptional atlas reveals resident progenitor cell niche functions in TMJ disc development and injury

The biological characteristics of the temporomandibular joint disc involve complex cellular network in cell identity and extracellular matrix composition to modulate jaw function. The lack of a detailed characterization of the network severely limits the development of targeted therapies for temporomandibular joint-related diseases. Here we profiled single-cell transcriptomes of disc cells from mice at different postnatal stages, finding that the fibroblast population could be divided into chondrogenic and non-chondrogenic clusters. We also find that the resident mural cell population is the source of disc progenitors, characterized by ubiquitously active expression of the NOTCH3 and THY1 pathways. Lineage tracing reveals that Myh11+ mural cells coordinate angiogenesis during disc injury but lost their progenitor characteristics and ultimately become Sfrp2+ non-chondrogenic fibroblasts instead of Chad+ chondrogenic fibroblasts. Overall, we reveal multiple insights into the coordinated development of disc cells and are the first to describe the resident mural cell progenitor during disc injury.

Thyroid hormone action controls multiple components of cell junctions at the ventricular zone in the newborn rat brain.

Thyroid hormone (TH) action controls brain development in a spatiotemporal manner. Previously, we demonstrated that perinatal hypothyroidism led to formation of a periventricular heterotopia in developing rats. This heterotopia occurs in the posterior telencephalon, and its formation was preceded by loss of radial glia cell polarity. As radial glia mediate cell migration and originate in a progenitor cell niche called the ventricular zone (VZ), we hypothesized that TH action may control cell signaling in this region. Here we addressed this hypothesis by employing laser capture microdissection and RNA-Seq to evaluate the VZ during a known period of TH sensitivity. Pregnant rats were exposed to a low dose of propylthiouracil (PTU, 0.0003%) through the drinking water during pregnancy and lactation. Dam and pup THs were quantified postnatally and RNA-Seq of the VZ performed in neonates. The PTU exposure resulted in a modest increase in maternal thyroid stimulating hormone and reduced thyroxine (T4). Exposed neonates exhibited hypothyroidism and T4 and triiodothyronine (T3) were also reduced in the telencephalon. RNA-Seq identified 358 differentially expressed genes in microdissected VZ cells of hypothyroid neonates as compared to controls (q-values ≤0.05). Pathway analyses showed processes like maintenance of the extracellular matrix and cytoskeleton, cell adhesion, and cell migration were significantly affected by hypothyroidism. Immunofluorescence also demonstrated that collagen IV, F-actin, radial glia, and adhesion proteins were reduced in the VZ. Immunohistochemistry of integrin αvβ3 and isoforms of both thyroid receptors (TRα/TRβ) showed highly overlapping expression patterns, including enrichment in the VZ. Taken together, our results show that TH action targets multiple components of cell junctions in the VZ, and this may be mediated by both genomic and nongenomic mechanisms. Surprisingly, this work also suggests that the blood-brain and blood-cerebrospinal fluid barriers may also be affected in hypothyroid newborns.

The tendon interfascicular basement membrane provides a vascular niche for CD146+ cell subpopulations.

Introduction: The interfascicular matrix (IFM; also known as the endotenon) is critical to the mechanical adaptations and response to load in energy-storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT). We hypothesized that the IFM is a tendon progenitor cell niche housing an exclusive cell subpopulation. Methods: Immunolabelling of equine superficial digital flexor tendon was used to identify the interfascicular matrix niche, localising expression patterns of CD31 (endothelial cells), Desmin (smooth muscle cells and pericytes), CD146 (interfascicular matrix cells) and LAMA4 (interfascicular matrix basement membrane marker). Magnetic-activated cell sorting was employed to isolate and compare in vitro properties of CD146+ and CD146- subpopulations. Results: Labelling for CD146 using standard histological and 3D imaging of large intact 3D segments revealed an exclusive interfascicular cell subpopulation that resides in proximity to a basal lamina which forms extensive, interconnected vascular networks. Isolated CD146+ cells exhibited limited mineralisation (osteogenesis) and lipid production (adipogenesis). Discussion: This study demonstrates that the interfascicular matrix is a unique tendon cell niche, containing a vascular-rich network of basement membrane, CD31+ endothelial cells, Desmin+ mural cells, and CD146+ cell populations that are likely essential to tendon structure and/or function. Contrary to our hypothesis, interfascicular CD146+ subpopulations did not exhibit stem cell-like phenotypes. Instead, our results indicate CD146 as a pan-vascular marker within the tendon interfascicular matrix. Together with previous work demonstrating that endogenous tendon CD146+ cells migrate to sites of injury, our data suggest that their mobilisation to promote intrinsic repair involves changes in their relationships with local interfascicular matrix vascular and basement membrane constituents.

ODP305 Differentiation Potential of Marginal Layer-Derived Adult Pituitary Stem/Progenitor Cells into Vascular Endothelial Cells and Pericytes in Mice

Abstract The anterior lobe (AL) of the pituitary gland consists five types of hormone-producing cells and fenestrated sinusoids (i. e., vascular endothelial cells and pericytes) that cooperate to maintain the physiological function of the gland. In addition, the population of cells located in pituitary stem/ progenitor cell niche, the marginal layer, which faces the residual lumen of the Rathke's pouch, serve to renew pituitary cells throughout life. We recently established a protocol for isolating marginal layer-derived adult pituitary stem/progenitor cells (MPSCs) by using differences in cell adhesion (1), and then showed differentiation potential into hormone-producing cells by subjecting MPSCs to three-dimensional culture. However, it is unclear whether MPSCs are capable of differentiating into all types of cells that constitute the pituitary gland. In this study, we performed DNA microarray analysis in MPSCs, which were proliferated by two-dimensional (2D) culture, for studying comprehensively gene expression profiles. Next, we investigated what kind of cells MPSCs would differentiate into. Comparing gene expression profiles of the AL and MPSCs, in MPSCs, 1776 genes were increased more than 4-fold and 1715 genes were decreased 4-fold compared to the AL (p<0. 05, respectively). The upregulated genes included pituitary stem/progenitor cell markers (ex. Sox2, Sox9, Klf4, Prrx1), niche markers (ex. Cdh1, Krt8), and cell surface antigens (ex. Cd9, Cd34, Cd44). On the other hand, most of the downregulated genes were differentiation markers (ex. Pou1f1, Gata2, Tbx19, Kdr). Next, immunofluorescent analysis revealed that most of the MPSCs were positive for CD34, the hematopoietic stem cell marker. After further 2D culture of MPSCs for 5-7days, vascular endothelial marker PECAM1-positive cells were detected by immunofluorescent analysis, but pericyte marker CSPG4-positive cell not. When TGFβ was applied to 2D culture of MPSCs, CSPG4-positive cells were detected instead of PECAM1-positive cells. Expression levels of Pecam1and Cspg4 were also examined by qPCR analysis, which corresponded to the results of immunofluorescent analysis. On the other hand, expression levels of anterior hormone genes were almost unchanged. These results have demonstrated that MPSCs could differentiate into vascular endothelial cells and pericytes. The present study suggests that pituitary stem/progenitor cells, which derived from the marginal layer, are a source of not only hormone-producing cells but also cells that constitute blood vessels. Reference: (1) Shintani and Higuchi, Stem Cell Res. 2021 Feb 2;52: 102223. Presentation: No date and time listed

MSC and HSPC Coculture: Mimicking Ex Vivo Bone Marrow Niche.

Mesenchymal stromal cells (MSCs) are the crucial component of the hematopoietic stem and progenitor cell (HSPC) niche in the bone marrow. Therefore, an ex vivo culture system that recapitulates the marrow microenvironment is important to understanding the niche's regulatory role on HSPC function and improving ex vivo HSPC expansion for clinical transplantation. Herein, a procedure for ex vivo expansion of MSCs from human bone marrow cells and their identification and characterization is described. In addition, a protocol for MSC and HSPC coculture assay is presented. This MSC-HSPC coculture assay can be used for ex vivo expansion of HSPC. Furthermore, this assay is also useful for qualitative analysis of MSCs capable of supporting hematopoiesis.

HIF-1alpha-pathway activation in cholangiocytes of patients with biliary atresia: An immunohistochemical/molecular exploratory study

BackgroundBiliary atresia is a neonatal disease characterized by choledochal obstruction and progressive cholangiopathy requiring liver transplantation in most patients. Hypoxia-ischemia affecting the biliary epithelium may lead to biliary obstruction. We hypothesized that ischemic cholangiopathy involving disruption of the peribiliary vascular plexus could act as a triggering event in biliary atresia pathogenesis. MethodsLiver and porta hepatis paraffin-embedded samples of patients with biliary atresia or intrahepatic neonatal cholestasis (controls) were immunohistochemically evaluated for HIF-1alpha-nuclear signals. Frozen histological samples were analyzed for gene expression in molecular profiles associated with hypoxia-ischemia. Prospective clinical-laboratory and histopathological data of biliary atresia patients and controls were reviewed. ResultsImmunohistochemical HIF-1alpha signals localized to cholangiocytes were detected exclusively in liver specimens from biliary atresia patients. In 37.5% of liver specimens, HIF-1alpha signals were observed in biliary structures involving progenitor cell niches and peribiliary vascular plexus. HIF-1alpha signals were also detected in biliary remnants of 81.8% of porta hepatis specimens. Increased gene expression of molecules linked to REDOX status, biliary proliferation, and angiogenesis was identified in biliary atresia liver specimens. In addition, there was a trend towards decreased GSR expression levels in the HIF-1alpha-positive group compared to the HIF-1alpha-negative group. ConclusionActivation of the HIF-1alpha pathway may be associated with the pathogenesis of biliary atresia, and additional studies are necessary to confirm the significance of this finding. Ischemic cholangiopathy and REDOX status disturbance are putative explanations for HIF-1alpha activation. These findings may give rise to novel lines of clinical and therapeutic investigation in the BA field.

THU541 - Wnt as activator of regeneration in cirrhosis of human liver: tissue collapse brings the Wnt source in hepatic veins to the site of the progenitor cell niche

THU541 - Wnt as activator of regeneration in cirrhosis of human liver: tissue collapse brings the Wnt source in hepatic veins to the site of the progenitor cell niche

The long and winding road: homeostatic and disordered haematopoietic microenvironmental niches: a narrative review.

ABSTRACTHaematopoietic microenvironmental niches have been described as the ‘gatekeepers’ for the blood and immune systems. These niches change during ontogeny, with the bone marrow becoming the predominant site of haematopoiesis in post-natal life under steady state conditions. To determine the structure and function of different haematopoietic microenvironmental niches, it is essential to clearly define specific haematopoietic stem and progenitor cell subsets during ontogeny and to understand their temporal appearance and anatomical positioning. A variety of haematopoietic and non-haematopoietic cells contribute to haematopoietic stem and progenitor cell niches. The latter is reported to include endothelial cells and mesenchymal stromal cells (MSCs), skeletal stem cells and/or C-X-C motif chemokine ligand 12-abundant-reticular cell populations, which form crucial components of these microenvironments under homeostatic conditions. Dysregulation or deterioration of such cells contributes to significant clinical disorders and diseases worldwide and is associated with the ageing process. A critical appraisal of these issues and of the roles of MSC/C-X-C motif chemokine ligand 12-abundant-reticular cells and the more recently identified skeletal stem cell subsets in bone marrow haematopoietic niche function under homeostatic conditions and during ageing will form the basis of this research review. In the context of haematopoiesis, clinical translation will deal with lessons learned from the vast experience garnered from the development and use of MSC therapies to treat graft versus host disease in the context of allogeneic haematopoietic transplants, the recent application of these MSC therapies to treating emerging and severe coronavirus disease 2019 (COVID-19) infections, and, given that skeletal stem cell ageing is one proposed driver for haematopoietic ageing, the potential contributions of these stem cells to haematopoiesis in healthy bone marrow and the benefits and challenges of using this knowledge for rejuvenating the age-compromised bone marrow haematopoietic niches and restoring haematopoiesis.