Expression Of Progenitor Cell Markers Research Articles

Abstract 12723: Smooth Muscle-Derived CD34 + Cells After Vascular Injury Contribute to Vascular Remodeling

Introduction: Vascular remodeling after injury participates in several atherosclerotic cardiovascular diseases. Vascular progenitors are thought to play vital roles in vascular remodeling, while the specific contribution of newly-generated progenitors from non-progenitors after injury is neglected. Methods: We generated a Cd34 lineage tracing system and marked newly-generated CD34 + cells in vascular injury models. Single-cell transcriptomes revealed cellular changes of CD34 + cells during vascular remodeling. To explore the cell origin of these CD34 + cells, we applied bone marrow (BM) transplantation, pseudo-time analysis of femoral artery cells, and further generated a smooth muscle (SM) lineage tracing system. To examine the role of Doublecortin Like Kinase 1 (DCLK1) in SM-derived CD34 + cell generation, overexpression of DCLK1 in primary VSMCs and genetic knockout of DCLK1 in SM lineage tracing system was performed. Results: A cluster of CD34 + cells was identified in femoral artery wire injury, which were CD34 negative before injury but CD34 positive after injury. These newly-generated CD34 + cells co-expressed VSMC or immune cell markers in neointima. BM transplantation clarified that vascular resident newly-generated CD34 + cells expressed VSMC makers or CD68, while BM CD34 + cells turned to immune cells but not VSMCs in neointima. These non-BM newly-generated CD34 + cells partly derived from VSMCs after injury. DCLK1 expression increased along the VSMCs-to-CD34 + cells transition. DCLK1 overexpression in VSMCs increased progenitor cell marker expression and decreased contractile VSMC marker expression. Genetic depletion of DCLK1 in VSMCs impaired SM-derived CD34 + cell generation during vascular remodeling. Conclusions: A novel cluster ‘SM-derived CD34 + cells’ was defined after vascular injury. These findings enriched our knowledge in pathological process of vascular remodeling and provided an avenue for future clinical interventions.

Epithelial plasticity enhances regeneration of committed taste receptor cells following nerve injury

Taste receptor cells are taste bud epithelial cells that are dependent upon the innervating nerve for continuous renewal and are maintained by resident tissue stem/progenitor cells. Transection of the innervating nerve causes degeneration of taste buds and taste receptor cells. However, a subset of the taste receptor cells is maintained without nerve contact after glossopharyngeal nerve transection in the circumvallate papilla in adult mice. Here, we revealed that injury caused by glossopharyngeal nerve transection triggers the remaining differentiated K8-positive taste receptor cells to dedifferentiate and acquire transient progenitor cell-like states during regeneration. Dedifferentiated taste receptor cells proliferate, express progenitor cell markers (K14, Sox2, PCNA) and form organoids in vitro. These data indicate that differentiated taste receptor cells can enter the cell cycle, acquire stemness, and participate in taste bud regeneration. We propose that dedifferentiated taste receptor cells in combination with stem/progenitor cells enhance the regeneration of taste buds following nerve injury.

SRSF10 is essential for progenitor spermatogonia expansion by regulating alternative splicing.

Alternative splicing expands the transcriptome and proteome complexity and plays essential roles in tissue development and human diseases. However, how alternative splicing regulates spermatogenesis remains largely unknown. Here, using a germ cell-specific knockout mouse model, we demonstrated that the splicing factor Srsf10 is essential for spermatogenesis and male fertility. In the absence of SRSF10, spermatogonial stem cells can be formed, but the expansion of Promyelocytic Leukemia Zinc Finger (PLZF)-positive undifferentiated progenitors was impaired, followed by the failure of spermatogonia differentiation (marked by KIT expression) and meiosis initiation. This was further evidenced by the decreased expression of progenitor cell markers in bulk RNA-seq, and much less progenitor and differentiating spermatogonia in single-cell RNA-seq data. Notably, SRSF10 directly binds thousands of genes in isolated THY+ spermatogonia, and Srsf10 depletion disturbed the alternative splicing of genes that are preferentially associated with germ cell development, cell cycle, and chromosome segregation, including Nasp, Bclaf1, Rif1, Dazl, Kit, Ret, and Sycp1. These data suggest that SRSF10 is critical for the expansion of undifferentiated progenitors by regulating alternative splicing, expanding our understanding of the mechanism underlying spermatogenesis.

Mature rat hepatocyte dedifferentiation into long lived proliferating hepatic progenitor cells

Objective: to obtain long-lived proliferating cells with progenitor features by dedifferentiation of mature rat hepatocytes using combinations of small molecules.Materials and Methods. Hepatocytes isolated from rat liver by perfusion were cultured in the presence of a cocktail of three small molecules – Wnt signaling pathway activator (CHIR99021), TGF-β inhibitors (A83-01) and ROCK kinase (Y27632). The morphological characteristics and growth features of the culture were assessed using fluorescence and phase-contrast microscopy during cell culture. Cell proliferative activity was analyzed using real-time time-lapse imaging. The expression of surface and intracellular markers was analyzed using flow cytometry and high-resolution fluorescence microscopy.Results. Using a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, long-lived proliferating cells that express progenitor cell markers, such as α-fetoprotein and HNF4α, were obtained from mature rat hepatocytes. The cells had hepatocyte-like morphology and formed discrete clusters of proliferating cells, forming a single cell layer during culturing. Removal of the small molecules from the medium led to expansion of fibroblast-like cells and elimination of potentially progenitor hepatocyte-like cells.Conclusion. Proliferating progenitor cells can be obtained by dedifferentiation of mature hepatocytes.

Reduced adult neurogenesis is associated with increased macrophages in the subependymal zone in schizophrenia.

Neural stem cells in the human subependymal zone (SEZ) generate neuronal progenitor cells that can differentiate and integrate as inhibitory interneurons into cortical and subcortical brain regions; yet the extent of adult neurogenesis remains unexplored in schizophrenia and bipolar disorder. We verified the existence of neurogenesis across the lifespan by chartering transcriptional alterations (2 days-103 years, n = 70) and identifying cells indicative of different stages of neurogenesis in the human SEZ. Expression of most neural stem and neuronal progenitor cell markers decreased during the first postnatal years and remained stable from childhood into ageing. We next discovered reduced neural stem and neuronal progenitor cell marker expression in the adult SEZ in schizophrenia and bipolar disorder compared to controls (n = 29-32 per group). RNA sequencing identified increased expression of the macrophage marker CD163 as the most significant molecular change in schizophrenia. CD163+ macrophages, which were localised along blood vessels and in the parenchyma within 10 µm of neural stem and progenitor cells, had increased density in schizophrenia but not in bipolar disorder. Macrophage marker expression negatively correlated with neuronal progenitor marker expression in schizophrenia but not in controls or bipolar disorder. Reduced neurogenesis and increased macrophage marker expression were also associated with polygenic risk for schizophrenia. Our results support that the human SEZ retains the capacity to generate neuronal progenitor cells throughout life, although this capacity is limited in schizophrenia and bipolar disorder. The increase in macrophages in schizophrenia but not in bipolar disorder indicates that immune cells may impair neurogenesis in the adult SEZ in a disease-specific manner.

Zbp1-positive cells are osteogenic progenitors in periodontal ligament

Periodontal ligament (PDL) possesses a stem/progenitor population to maintain the homeostasis of periodontal tissue. However, transcription factors that regulate this population have not yet been identified. Thus, we aimed to identify a molecule related to the osteogenic differentiation of PDL progenitors using a single cell-based strategy in this study. We first devised a new protocol to isolate PDL cells from the surface of adult murine molars and established 35 new single cell-derived clones from the PDL explant. Among these clones, six clones with high (high clones, n = 3) and low (low clones, n = 3) osteogenic potential were selected. Despite a clear difference in the osteogenic potential of these clones, no significant differences in their cell morphology, progenitor cell marker expression, alkaline phosphatase activity, proliferation rate, and differentiation-related gene and protein expression were observed. RNA-seq analysis of these clones revealed that Z-DNA binding protein-1 (Zbp1) was significantly expressed in the high osteogenic clones, indicating that Zbp1 could be a possible marker and regulator of the osteogenic differentiation of PDL progenitor cells. Zbp1-positive cells were distributed sparsely throughout the PDL. In vitro Zbp1 expression in the PDL clones remained at a high level during osteogenic differentiation. The CRISPR/Cas9 mediated Zbp1 knockout in the high clones resulted in a delay in cell differentiation. On the other hand, Zbp1 overexpression in the low clones promoted cell differentiation. These findings suggested that Zbp1 marked the PDL progenitors with high osteogenic potential and promoted their osteogenic differentiation. Clarifying the mechanism of differentiation of PDL cells by Zbp1 and other factors in future studies will facilitate a better understanding of periodontal tissue homeostasis and repair, possibly leading to the development of novel therapeutic measures.

Chemical conversion of aged hepatocytes into bipotent liver progenitor cells.

In the aging society, understanding the influence of hepatocyte age on hepatocyte donation may inform efforts to expand alternative cell sources to mitigate liver donor shortage. A combination of the molecules Y27632, A-83-01, and CHIR99021 has been used to reprogram rodent young hepatocytes into chemically induced liver progenitor (CLiP) cells; however, whether it could also reprogram aged hepatocytes has not yet been elucidated. Primary hepatocytes were isolated from aged and young donor rats, respectively. Hepatic histological changes were evaluated. Differences in gene expression in hepatocytes were identified. The in vitro reprogramming plasticity of hepatocytes as evidenced by CLiP conversion and the hepatocyte and cholangiocyte maturation capacity of reprogrammed CLIPs were analyzed. The effect of hepatocyte growth factor (HGF) on cell propagation was also investigated. The histological findings revealed ongoing liver damage with inflammation, fibrosis, senescence, and ductular reaction in aged livers. Microarray analysis showed altered gene expression profiles in hepatocytes from aged donors, especially with regard to metabolic pathways. Aged hepatocytes could be converted into CLiPs (Aged-CLiPs) expressing progenitor cell markers, but with a relatively low proliferative rate compared with young hepatocytes. Aged-CLiPs possessed both hepatocyte and cholangiocyte maturation capacity. HGF facilitated CLiP conversion in aged hepatocytes, which was partly related to the activation of Erk1 and Akt1 signaling. Aged rat hepatocytes have retained reprogramming plasticity as evidenced by CLiP conversion in culture. HGF promoted proliferation and CLiP conversion in aged hepatocytes. Hepatocytes from aged donors may be used as an alternative cell source to mitigate donor shortage.

SURGICAL RETINAL EXPLANTS AS A SOURCE OF RETINAL PROGENITOR CELLS.

To describe the novel observation of spontaneously migrating retinal cells from living donor surgical retinal explants that express progenitor cell markers in the absence of exogenous growth factors. Surgical retinal explants were harvested from 5 consecutive patients undergoing 23 G pars plana vitrectomy for the management of rhegmatogenous detachment. During surgery, equatorial flap tears were trimmed with the vitreous cutter and aspirated. Excised tissue was then regurgitated into a syringe containing balanced salt solution and immediately transferred to tissue culture. Migrating cells subsequently underwent immunohistochemical staining and their characteristics were compared with those of a spontaneously immortalized Müller stem cell line. Spontaneously migrating cells were observed from samples taken from all 5 patients from Day 2 to 10 after transfer to culture. These cells were found to express embryonic cell markers, including paired box 6 (Pax6), sex-determining region Y-box 2 (Sox-2), nestin, cone-rod homeobox, and cyclin-dependent kinase inhibitor 1B (p27Kip1) as well as proteins consistent with early or retained differentiation down the Müller cell lineage, including glial fibrillary acidic protein and glutamine synthetase. After injury, the human equatorial retina is capable of spontaneously producing cells that demonstrate migration and that express progenitor cell markers. In addition, these cells express proteins consistent with Müller cell lineage. These initial observations support the assertion that the human retina may possess the potential for regeneration and that surgical retinal explants could also act as a ready source of retinal progenitor cells.

Noninvasive Electrical Stimulation Improves Photoreceptor Survival and Retinal Function in Mice with Inherited Photoreceptor Degeneration.

PurposeNeurons carry electrical signals and communicate via electrical activities. The therapeutic potential of electrical stimulation (ES) for the nervous system, including the retina, through improvement of cell survival and function has been noted. Here we investigated the neuroprotective and regenerative potential of ES in a mouse model of inherited retinal degeneration.MethodsRhodopsin-deficient (Rho−/−) mice received one or two sessions of transpalpebral ES or sham treatments for 7 consecutive days. Intraperitoneal injection of 5-ethynyl-2′-deoxyuridine was used to label proliferating cells. Weekly electroretinograms were performed to monitor retinal function. Retinal morphology, photoreceptor survival, and regeneration were evaluated in vivo using immunohistochemistry and genetic fate-mapping techniques. Müller cell (MC) cultures were employed to further define the optimal conditions of ES application.ResultsNoninvasive transpalpebral ES in Rho−/− mice improved photoreceptor survival and electroretinography function in vivo. ES also triggered residential retinal progenitor-like cells such as MCs to reenter the cell cycle, possibly producing new photoreceptors, as shown by immunohistochemistry and genetic fate-mapping techniques. ES directly stimulated cell proliferation and the expression of progenitor cell markers in MC cultures, at least partially through bFGF signaling.ConclusionsOur study showed that transpalpebral ES improved photoreceptor survival and retinal function and induced the proliferation, probably photoreceptor regeneration, of MCs; this occurs via stimulation of the bFGF pathways. These results suggest the exciting possibility of applying noninvasive ES as a versatile tool for preventing photoreceptor loss and mobilizing endogenous progenitors for reversing vision loss in patients with photoreceptor degeneration.

Electrical Stimulation Induces Retinal Müller Cell Proliferation and Their Progenitor Cell Potential.

Non-invasive electrical stimulation (ES) is increasingly applied to improve vision in untreatable eye conditions, such as retinitis pigmentosa and age-related macular degeneration. Our previous study suggested that ES promoted retinal function and the proliferation of progenitor-like glial cells in mice with inherited photoreceptor degeneration; however, the underlying mechanism remains obscure. Müller cells (MCs) are thought to be dormant residential progenitor cells that possess a high potential for retinal neuron repair and functional plasticity. Here, we showed that ES with a ramp waveform of 20 Hz and 300 µA of current was effective at inducing mouse MC proliferation and enhancing their expression of progenitor cell markers, such as Crx (cone–rod homeobox) and Wnt7, as well as their production of trophic factors, including ciliary neurotrophic factor. RNA sequencing revealed that calcium signaling pathway activation was a key event, with a false discovery rate of 5.33 × 10−8 (p = 1.78 × 10−10) in ES-mediated gene profiling changes. Moreover, the calcium channel blocker, nifedipine, abolished the observed effects of ES on MC proliferation and progenitor cell gene induction, supporting a central role of ES-induced Ca2+ signaling in the MC changes. Our results suggest that low-current ES may present a convenient tool for manipulating MC behavior toward neuroregeneration and repair.

Noninvasive prediction of HCC with progenitor phenotype based on gadoxetic acid-enhanced MRI.

To explore the noninvasive prediction of hepatocellular carcinoma (HCC) with progenitor phenotype based on gadoxetic acid-enhanced magnetic resonance imaging (MRI). This retrospective study included 115 surgery-proven HCCs with preoperative gadoxetic acid-enhanced MRI from August 2015 to September 2018. Image features were reviewed. Quantitative image analysis was performed using histogram analysis. HCC with progenitor phenotype was defined as positive for either cytokeratin 19 (CK19) or epithelial cell adhesion molecule (EpCAM) expression. Statistically significant variables for identifying HCCs with progenitor phenotype were determined at multivariate analyses. ROC analyses were used to determined cutoff values and the diagnostic performance of significant variables and combinations. Prediction nomogram was constructed based on multivariate analysis. At multivariate regression analyses, AFP ≥ 155.25ng/mL (p < 0.001), skewness on T2WI ≤ 1.10 (p = 0.024), uniformity on pre-T1WI ≤ 0.91 (p = 0.024), irregular tumor margin (p = 0.006), targetoid appearance (p = 0.001), and the absence of mosaic architecture (p = 0.014) were significant predictors of HCCs expressing progenitor cell markers. Combing any three of those significant variables, it provides a diagnostic accuracy of 0.86 (95% CI 0.78-0.92) with sensitivity of 0.97 (95% CI 0.86-1.00), and specificity of 0.74 (95% CI 0.63-0.83). The C-index of the regression coefficient-based nomogram was 0.94 (95% CI 0.91-0.98). Noninvasive prediction of HCCs with progenitor phenotype can be achieved with high accuracy by integrated interpretation of biochemical and radiological information, representing a handy tool for precise patient management and the prediction of prognosis. • Qualitative image features of irregular tumor margin, targetoid appearance, and the absence of mosaic architecture are significant predictors of hepatocellular carcinoma with progenitor phenotype. • Quantitative analyses using whole-lesion histogram analysis provides additional information for the prediction of hepatocellular carcinoma with progenitor phenotype. • Noninvasive prediction of hepatocellular carcinoma with progenitor phenotype can be achieved with high accuracy by integrated interpretation of clinical information and qualitative and quantitative imaging analyses.

Transcriptomic Dissection of Hepatocyte Heterogeneity: Linking Ploidy, Zonation, and Stem/Progenitor Cell Characteristics.

Background & AimsThere is a long-standing debate regarding the biological significance of polyploidy in hepatocytes. Recent studies have provided increasing evidence that hepatocytes with different ploidy statuses behave differently in a context-dependent manner (eg, susceptibility to oncogenesis, regenerative ability after injury, and in vitro proliferative capacity). However, their overall transcriptomic differences in a physiological context is not known.MethodsBy using microarray transcriptome analysis, we investigated the heterogeneity of hepatocyte populations with different ploidy statuses. Moreover, by using single-cell quantitative reverse-transcription polymerase chain reaction (scPCR) analysis, we investigated the intrapopulational transcriptome heterogeneity of 2c and 4c hepatocytes.ResultsMicroarray analysis showed that cell cycle–related genes were enriched in 8c hepatocytes, which is in line with the established notion that polyploidy is formed via cell division failure. Surprisingly, in contrast to the general consensus that 2c hepatocytes reside in the periportal region, in our bulk transcriptome and scPCR analyses, the 2c hepatocytes consistently showed pericentral hepatocyte-enriched characteristics. In addition, scPCR analysis identified a subpopulation within the 2c hepatocytes that co-express the liver progenitor cell markers Axin2, Prom1, and Lgr5, implying the potential biological relevance of this subpopulation.ConclusionsThis study provides new insights into hepatocyte heterogeneity, namely 2c hepatocytes are preferentially localized to the pericentral region, and a subpopulation of 2c hepatocytes show liver progenitor cell–like features in terms of liver progenitor cell marker expression (Axin2, Prom1, and Lgr5).

SAT-172 Continuous, Prolonged Treatment with the Oligonucleotide IMT504 in Diabetic NOD Mice Greatly Improves All Parameters of the Diabetic Condition

We have shown that the immunomodulatory oligonucleotide IMT504 (IMT) induces a marked recovery of single-dose streptozotocin (STZ)-induced toxic diabetes in rats that correlates with early expression of progenitor cell markers but without altering immune parameters. IMT also improves the diabetic condition in an immunodependent diabetes model induced by multiple low doses of STZ in mice, diminishing glycemia and reducing leukocyte islet infiltration. Besides, a short-term IMT treatment induces an early recovery on glucose homeostasis and insulitis on a spontaneous model of type I diabetes, NOD mice. Here, we evaluated the effects of a continuous chronic IMT treatment in NOD diabetic mice, more similar to what could eventually be used in humans Diabetic female NOD mice (non-fasted glycemia (Gly) levels between 250-350 mg/dl) were sc implanted with constant drug release pumps (Alzet osmotic pumps) with a capacity for 28 days, loaded with IMT (total dose released per day: 20 mg/kg BW) or saline (diabetic control: DC) (day1). Gly and body weight (BW) were determined once a week during the treatment. The weekly food intake was also determined in weeks 1 and 4. On day 21, the intraperitoneal glucose tolerance test (ipGTT) and insulin secretion test (IST) were performed. On day 28, fasted mice were sacrificed, blood samples and pancreases collected for hormonal determinations and histological studies respectively. We observed that 22% of NOD mice showed spontaneous reversion of the diabetic condition whereas IMT treatment ameliorated Gly in 78% of mice (X2: p<0.05). IMT-treated animals quickly attained normal Gly, which was maintained until the end of the experiment [ANOVA, interaction p<0.001; Gly (mg/dl) day 1= DC: 318±10, IMT: 301±12 vs day 7= DC: 330±72, IMT: 174±17; p<0.05 and vs day 28: DC: 540±23, IMT: 151±17; p<0.01]. IMT induced a significant recovery on glucose clearence [GTT: ANOVA: interaction p<0.02, 0 min= DC: 390±62 vs IMT: 141±23, p<0.002; 30 min= DC: 581±56 vs IMT: 239±32, p<0.004; 120 min DC: 422±53 vs IMT: 149±23, p<0.001] as well as on insulin secretion in response to the glucose overload [IST: ANOVA: interaction p<0.02; 0 min= DC: 0.16±0.14 vs IMT: 0.22±0.03, ns; 10 min= DC: 0.15±0.01 vs IMT: 0.33±0.05, p<0.04; 60 min DC: 0.15±0.02 vs IMT: 0.18±0.02, ns]. Body weights did not differ between groups. IMT treatment prevented food intake increase observed in DC group [ANOVA, interaction p<0.001; week 1= DC: 29.8±1.3 vs IMT: 26.0±1.4, ns; week 4= DC: 58.1±3.4 vs IMT: 29.0±1.3, p<0.001]. Besides, beta cell function was improved in IMT-treated animals [HOMA beta cell= DC: 12.3±6.3 vs IMT: 125±48.3, p<0.001]. These results demonstrate the effectiveness of prolonged and constant IMT treatment in promoting a significant improvement in terms of glycemic control and the diabetic condition in this model of type I diabetes. Fundings: CONICET, ANPCYT, UBA, Johnson&Johnson Arg, Fund R Barón, Fund Williams

Expression of Progenitor Cell Markers in the Glial-Like Cells of Epiretinal Membranes of Different Origins.

Purpose To investigate the expression of progenitor cell markers (Sox2, Nestin, and Pax2) in idiopathic epiretinal membranes (iERMs) and nonidiopathic epiretinal membranes (niERMs) in relation to glial cell marker expression. Methods ERMs were obtained from patients with iERMs and niERMs of different origins: proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and uveitis. The membranes were studied by flat-mount or sectional immunohistochemistry for expression of progenitor cell markers as well as glial (GFAP) and proliferation (Ki-67) markers. Results Cells in the ERMs express strong GFAP, with strong Pax2 expression in the cell nuclei. Some of the GFAP-positive glial cells in all epiretinal membrane types colocalized with Sox2, Pax2, and Nestin. NiERMs are much more cellular than iERMs. Glial cells are more densely packed in all analyzed niERMs, whereas glial cells with long branches are found in the internal limiting membrane parts and the iERMs, which appear to form a local network by their processes. Conclusion The GFAP-positive glial cells in ERMs are not pure glial cells, and some of them express progenitor cell markers, which indicate that these cells may have potential for self-renewal and differentiation into more glial or neuroglial type of cells.

Visualization of global RNA synthesis in a human (mini-) organ in situ by click chemistry.

RNA synthesis can be detected by 5-ethynyl uridine (EU) incorporation and click chemistry. Despite identifying a fundamental functional process, this technique has yet to be widely applied to complex human tissue systems. By incorporating EU into human hair follicle (HF) organs cultured ex vivo, nascent RNA synthesis was detected in situ. EU differentially incorporated across the HF epithelium.Interestingly, RNA synthesis did not correlate with protein synthesis, proliferation or epithelial progenitor cell marker expression. By treating human HFs with the cytotoxic cell cycle inhibitor (R)-CR8, which inhibits transcriptional regulators CDK7 and CDK9, it was further shown that this technique can be used to sensitively detect changes in global RNA synthesis in situ. Together, this work delineates new insights into nascent RNA synthesis within a human (mini)- organ and describes a novel read-out parameter that will enrich future ex vivo human tissue research studies.

Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells

Understanding transcription factor (TF) regulation of limbal epithelial stem/progenitor cells (LEPCs) may aid in using non-ocular cells to regenerate the corneal surface. This study aimed to identify and characterize TF genes expressed specifically in LEPCs isolated from human donor eyes by laser capture microdissection. Using a profiling approach, preferential limbal expression was found for SoxE and SoxF genes, particularly for Sox9, which showed predominantly cytoplasmic localization in basal LEPCs and nuclear localization in suprabasal and corneal epithelial cells, indicating nucleocytoplasmic translocation and activation during LEPC proliferation and differentiation. Increased nuclear localization of Sox9 was also observed in activated LEPCs following clonal expansion and corneal epithelial wound healing. Knockdown of SOX9 expression in cultured LEPCs by RNAi led to reduced expression of progenitor cell markers, e.g. keratin 15, and increased expression of differentiation markers, e.g. keratin 3. Furthermore, SOX9 silencing significantly suppressed the proliferative capacity of LEPCs and reduced levels of glycogen synthase kinase 3 beta (GSK-3ß), a negative regulator of Wnt/ß-catenin signaling. Sox9 expression, in turn, was significantly suppressed by treatment of LEPCs with exogenous GSK-3ß inhibitors and enhanced by small molecule inhibitors of Wnt signaling. Our results suggest that Sox9 and Wnt/ß-catenin signaling cooperate in mutually repressive interactions to achieve a balance between quiescence, proliferation and differentiation of LEPCs in the limbal niche. Future molecular dissection of Sox9-Wnt interaction and mechanisms of nucleocytoplasmic shuttling of Sox9 may aid in improving the regenerative potential of LEPCs and the reprogramming of non-ocular cells for corneal surface regeneration.

Identification and In Vitro Expansion of Buccal Epithelial Cells

Ex vivo-expanded buccal mucosal epithelial (BME) cell transplantation has been used to reconstruct the ocular surface. Methods for enrichment and maintenance of BME progenitor cells in ex vivo cultures may improve the outcome of BME cell transplantation. However, the parameter of cell seeding density in this context has largely been neglected. This study investigates how varying cell seeding density influences BME cell proliferation and differentiation on tissue culture polystyrene (TCPS). The highest cell proliferation activity was seen when cells were seeded at 5×104 cells/cm2. Both below and above this density, the cell proliferation rate decreased sharply. Differential immunofluorescence analysis of surface markers associated with the BME progenitor cell population (p63, CK19, and ABCG2), the differentiated cell marker CK10 and connexin 50 (Cx50) revealed that the initial cell seeding density also significantly affected the progenitor cell marker expression profile. Hence, this study demonstrates that seeding density has a profound effect on the proliferation and differentiation of BME stem cells in vitro, and this is relevant to downstream cell therapy applications.

Liver damage, proliferation, and progenitor cell markers in experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a disease known to cause injury to multiple organs including the liver. Liver regeneration is essential for the recovery after NEC-induced liver injury. Our aim was to investigate hepatic proliferation and progenitor cell marker expression in experimental NEC. Following ethical approval (#32238), NEC was induced in mice by hypoxia, gavage feeding of hyperosmolar formula, and lipopolysaccharide. Breastfed pups were used as control. We analyzed serum ALT level, liver inflammatory cytokines, liver proliferation markers, and progenitor cell marker expression. Comparison was made between NEC and controls. Serum ALT level was higher in NEC (p<0.05). The mRNA expression of inflammatory cytokines in the liver was also higher in NEC (IL6: p<0.05, TNF-α: p<0.01). Conversely, mRNA expression of proliferation markers in the liver was lower in NEC (Ki67; p<0.01, PCNA: p<0.01). LGR5 expression was also significantly decreased in NEC as demonstrated by mRNA (p<0.05) and protein (p<0.01) levels. Inflammatory injury was present in the liver during experimental NEC. Proliferation and LGR5 expression were impaired in the NEC liver. Modulation of progenitor cell expressing LGR5 may result in stimulation of liver regeneration in NEC-induced liver injury and improved clinical outcome. Level IV.

Increased caveolin-1 in intervertebral disc degeneration facilitates repair

BackgroundPreceding intervertebral disc (IVD) degeneration, the cell phenotype in the nucleus pulposus (NP) shifts from notochordal cells (NCs) to chondrocyte-like cells (CLCs). Microarray analysis showed a correlation between caveolin-1 expression and the phenotypic transition of NCs to CLCs. With a clinical directive in mind, the aim of this study was to determine the role of caveolin-1 in IVD degeneration. As a scaffolding protein, caveolin-1 influences several signaling pathways, and transforming growth factor (TGF)-β receptors have been demonstrated to colocalize with caveolin-1. Therefore, the hypothesis of this study was that caveolin-1 facilitates repair by enhancing TGF-β signaling in the IVD.MethodsProtein expression (caveolin-1, apoptosis, progenitor cell markers, extracellular matrix, and phosphorylated Smad2 [pSmad2]) was determined in IVDs of wild-type (WT) and caveolin-1-null mice and canine IVDs of different degeneration grades (immunofluorescence, immunohistochemistry, and TUNEL assay). Canine/human CLC microaggregates were treated with chondrogenic medium alone or in combination with caveolin-1 scaffolding domain (CSD) peptide and/or caveolin-1 silencing RNA. After 28 days, gene and protein expression profiles were determined.ResultsThe NP of WT mice was rich in viable NCs, whereas the NP of caveolin-1-null mice contained more collagen-rich extracellular matrix and fewer cells, together with increased progenitor cell marker expression, pSmad2 TGF-β signaling, and high apoptotic activity. During canine IVD degeneration, caveolin-1 expression and apoptotic activity increased. In vitro caveolin-1 silencing decreased the CLC microaggregate glycosaminoglycan (GAG) content, which could be rescued by CSD treatment. Furthermore, CSD increased TGF-β/pSmad2 signaling at gene and protein expression levels and enhanced the anabolic effects of TGF-β1, reflected in increased extracellular matrix deposition by the CLCs.ConclusionsCaveolin-1 plays a role in preservation of the NC phenotype. Additionally, it may be related to CLC apoptosis, given its increased expression in degenerated IVDs. Nevertheless, CSD enhanced CLC GAG deposition in vitro, and hence the increased caveolin-1 expression during IVD degeneration may also facilitate an ultimate attempt at repair. Further studies are needed to investigate how caveolin-1 modifies other signaling pathways and facilitates IVD repair.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-0960-y) contains supplementary material, which is available to authorized users.

The Characteristics Variation of Hepatic Progenitors after TGF-β1-Induced Transition and EGF-Induced Reversion.

Profibrogenesis cytokine, transforming growth factor- (TGF-) β1, induces hepatic progenitors experiencing epithelial to mesenchymal transition (EMT) to matrix synthesis cells, even tumor initiating cells. Our previous data found that epidermal growth factor (EGF) blocks and reverses TGF-β1-induced transition. The aim of this study is to determine the characteristic changes of hepatic progenitors after TGF-β1-induced transition and EGF-induced reversion. Hepatic oval cells, rat hepatic progenitors, were isolated from rats fed a choline-deficient diet supplemented with ethionine. TGF-β1-containing medium was used for inducing EMT, while EGF-containing medium was used for reversing EMT. During TGF-β1-induced transition and EGF-induced reversion, hepatic oval cells sustained their progenitor cell marker expression, including α-fetoprotein, albumin, and cytokeratin-19. The proliferation ability and differentiation potential of these cells were suppressed by TGF-β1, while EGF resumed these capacities to the level similar to the control cells. RNA microarray analysis showed that most of the genes with significant changes after TGF-β1 incubation were recovered by EGF. Signal pathway analysis revealed that TGF-β1 impaired the pathways of cell cycle and cytochrome P450 detoxification, and EGF reverted TGF-β1 effects through activating MAPK and PI3K-Akt pathway. EGF reverses the characteristics impaired by TGF-β1 in hepatic oval cells, serving as a protective cytokine to hepatic progenitors.