MicroRNA 219-5p inhibits alveolarization by reducing platelet derived growth factor receptor-alpha.

BACKGROUND: MicroRNA (miR) are small conserved RNA that regulate gene expression post-transcription. Previous genome-wide analysis studies in preterm infants indicate that pathways of miR 219-5p are important in infants with Bronchopulmonary Dysplasia (BPD). METHODS: Here we report a prospective cohort study of extremely preterm neonates wherein infants diagnosed with severe BPD expressed increased airway miR-219-5p and decreased platelet derived growth factor receptor alpha (PDGFR-α), a target of mir-219-5p and a key regulator of alveolarization, compared to post-conception age-matched term infants. RESULTS: miR-219-5p was highly expressed in the pulmonary epithelial lining in lungs of infants with BPD by in situ hybridization of human infant lungs. In both in vitro and in vivo (mouse) models of BPD, miR-219-5p was increased on exposure to hyperoxia compared with the normoxia control, with a complementary decrease of PDGFR-α. To further confirm the target relationship between miR-219 and PDGFR-α, pulmonary epithelial cells (MLE12) and lung primary fibroblasts were treated with a mimic of miR-219-5p and a locked nucleic acid (LNA) based inhibitor of miR-219-5p. In comparison with the control group, the level of miR-219 increased significantly after miR-219 mimic treatment, while the level of PDGFR-α declined markedly. LNA exposure increased PDGFR-α. Moreover, in BPD mouse model, over-expression of miR-219-5p inhibited alveolar development, indicated by larger alveolar spaces accompanied by reduced septation. CONCLUSIONS: Taken together, our results demonstrate that increased miR-219-5p contributes to the pathogenesis of BPD by targeting and reducing PDGFR-α. The use of specific miRNA antagonists may be a therapeutic strategy for preventing the development of BPD.

Molecular and Genetic Evidence for the PDGFRα-Independent Population of Oligodendrocyte Progenitor Cells in the Developing Mouse Brain.

PDGFRα, specifically expressed by immature oligodendrocyte progenitor cells (OPCs) in the CNS, plays a critical role in OPC proliferation and migration. However, it has been uncertain whether all cells of oligodendrocyte lineage are derived from the PDGFRα-expressing OPCs. In the present study, we uncovered a PDGFRα-independent oligodendrocyte lineage in the developing cortex. This OPC subpopulation originates from the local ventricular/subventricular zone after birth and contributes to the earliest mature oligodendrocytes in the cortex. PDGFRα signaling does not regulate the generation and differentiation of cortical OPCs. Fate-mapping studies in the PDGFRαCreER; Sox10-GFP/tdTom double-transgenic mice of either sex have further corroborated the PDGFRα-independent oligodendrocyte lineage. This study provides additional missing genetic evidence for PDGFRα-independent oligodendrocyte lineage in the developing hindbrain.SIGNIFICANCE STATEMENT This is the first report of a subpopulation of oligodendrocyte lineage in the developing mouse cortex independent of PDGFRα signaling. These oligodendrocyte progenitor cells are generated from the local ventral ventricular zone/subventricular zone after birth, and contribute to the earliest mature oligodendrocytes in the cortex.

MiR-218 regulated cardiomyocyte differentiation and migration in mouse embryonic stem cells by targeting PDGFRα.

MicroRNAs (miRNAs) have been identified as key players in cardiogenesis and heart pathophysiological processes. However, many miRNAs are still not recognized for their roles in cardiomyocytes differentiation. In this study, we evaluated the effects of microRNA-218 (miR-218) in cardiomyocyte differentiation of the mouse embryonic stem cells (ESCs) in vitro. The percentage of the beating embryoid bodies (EBs) in miR-218 mimic-treated cells was reduced to 32% compared with miR-218 mimic negative control (56%) on day 5 + 3. The amplitude of the intracellular Ca2+ transients in the cardiomyocytes derived from ESCs was reduced upon miR-218 overexpression, followed by the decreased calcium-related proteins and cell junction proteins expressions. Besides, miR-218 expression in ESCs was related to the directional spreading ability of EBs during differentiation. The increased expression of miR-218 could promote the migration of ESCs in vitro, while the decreased expression of miR-218 could inhibit the migration by the transwell experiment. Meanwhile, miR-218 could regulate cell migration-related proteins Cdc42 and Rac1. Platelet-derived growth factor receptor α (PDGFRα) was further confirmed to be a direct target of miR-218 both physically and functionally by dual-luciferase reporter assay. Our data further described that overexpression of PDGFRα rescued the miR-218-mediated inhibition of cardiomyocyte differentiation and restored the miR-218-mediated promotion of cell migration. In conclusion, miR-218 was demonstrated to exert an inhibitory function and promoted cell migration via targeting PDGFRα during cardiomyocyte differentiation from ESCs. The current study revealed the role of miR-218 and may provide an important hint for cardiomyocyte differentiation of ESCs and induced pluripotent stem cells.

M1/M2 Macrophages Play Different Roles in Adipogenic Differentiation of PDGFRα+ Preadipocytes In Vitro.

BACKGROUND: PGGFRα+ preadipocytes are the major subpopulations that can regenerate into adipocytes. Two different types of macrophages exist in the fat tissue: the classically activated macrophage (M1) and the alternatively activated macrophage (M2). In this study, we investigated whether M1/M2 macrophages play distinct roles in adipogenic differentiation of PDGFRα+ preadipocytes. METHODS: Mouse preadipocytes and macrophages were isolated from C57BL/6 male mice of 6-8 weeks. The culture supernate of M1 and M2 macrophages was collected and co-cultured with the PDGFRα+ preadipocytes. After 3 days, Oil Red O staining was used to evaluate to adipogenic differentiation of PDGFRα+ preadipocytes and the expression of adipogenic-related transcription factors (C/EBP-α, PPARγ) were also tested. RESULTS: The results showed that when cultured in vitro, M1 macrophages could significantly suppress the adipogenesis of PDGFRα+ preadipocytes as well as the C/EBP-α and PPARγ expression, but M2 macrophages did not have significant influence on the adipogenesis of PDGFRα+ preadipocytes nor on C/EBP-α and PPARγ expression compared with the control group. CONCLUSIONS: M1 macrophages significantly suppress PDGFRα+ preadipocyte adipogenesis which provides a possible way to improve adipogenesis and fat retention after fat-free grafting by mitigating acute inflammation and manipulating M1 macrophage levels. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Effects of FGFR Signaling on Cell Proliferation and Differentiation of Apert Dental Cells.

The Apert syndrome is a rare congenital disorder most often arising from S252W or P253R mutations in fibroblast growth factor receptor (FGFR2). Numerous studies have focused on the regulatory role of Apert FGFR2 signaling in bone formation, whereas its functional role in tooth development is largely unknown. To investigate the role of FGFR signaling in cell proliferation and odontogenic differentiation of human dental cells in vitro, we isolated dental pulp and enamel organ epithelia (EOE) tissues from an Apert patient carrying the S252W FGFR2 mutation. Apert primary pulp and EOE cells were established and shown to exhibit normal morphology and express alkaline phosphatase under differentiation conditions. Similar to control cells, Apert dental pulp and EOE cells expressed all FGFRs, with highest levels of FGFR1 followed by FGFR2 and low levels of FGFR3 and FGFR4. However, Apert cells had increased cell growth compared with control cells. Distinct from previous findings in osteoblast cells, gain-of-function S252W FGFR2 mutation did not upregulate the expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFRα), but elevated extracellular signal-regulated kinase (ERK) signaling in cells after EGF stimulation. Unexpectedly, there was little effect of the S252W mutation on odontogenic gene expression in dental pulp and EOE cells. However, after inhibition of total FGFR signaling or ERK signaling, the expression of odontogenic genes was upregulated in both dental cell types, indicating the negative effect of whole FGFR signaling on odontogenic differentiation. This study provides novel insights on FGFR signaling and a common Apert FGFR2 mutation in the regulation of odontogenic differentiation of dental mesenchymal and epithelial cells.

Pax3 and Tbx5 specify whether PDGFRα+ cells assume skeletal or cardiac muscle fate in differentiating embryonic stem cells.

Embryonic stem cells (ESCs) represent an ideal model to study how lineage decisions are established during embryonic development. Using a doxycycline-inducible mouse ESC line, we have previously shown that expression of the transcriptional activator Pax3 in early mesodermal cells leads to the robust generation of paraxial mesoderm progenitors that ultimately differentiate into skeletal muscle precursors. Here, we show that the ability of this transcription factor to induce the skeletal myogenic cell fate occurs at the expenses of the cardiac lineage. Our results show that the PDGFRα+FLK1--subfraction represents the main population affected by Pax3, through downregulation of several transcripts encoding for proteins involved in cardiac development. We demonstrate that although Nkx2-5, Tbx5, and Gata4 negatively affect Pax3 skeletal myogenic activity, the cardiac potential of embryoid body-derived cultures is restored solely by forced expression of Tbx5. Taking advantage of this model, we used an unbiased genome-wide approach to identify genes whose expression is rescued by Tbx5, and which could represent important regulators of cardiac development. These findings elucidate mechanisms regulating the commitment of mesodermal cells in the early embryo and identify the Tbx5 cardiac transcriptome.

Novel fusion between the breakpoint cluster region and platelet-derived growth factor receptor-alpha genes in a patient with chronic myeloid leukemia-like neoplasm: undetectable residual disease after imatinib therapy.

Rare patients suffering from myeloid neoplasms share clinical and cytological features indistinguishable from chronic myeloid leukemia (CML) but lack the BCR-ABL1 fusion gene. Several studies provide evidence that alterations in genes encoding tyrosine kinase receptors such as the platelet-derived growth factor receptor (PDGFR) may be involved in the pathogenesis of these disorders. Here we describe a patient with a rare CML-like disease in whom we identified a novel in-frame BCR-PDGFRA rearrangement joining BCR exon 17 to PDGFRA exon 13, resulting in overexpression of PDGFRA. The design of a specific quantitative PCR assay to monitor the molecular response during treatment with imatinib, a multitargeted tyrosine kinase inhibitor (TKI) with activity against ABL, c-Kit, and PDGFRA revealed an outstanding disease control with durably undetectable BCR-PDGFRA transcripts. Multiple TKIs are currently available yet with distinct target profiles; thus, accurate molecular diagnosis and monitoring tools are essential to establish tailored treatments and assess response to therapy in this type of rare hematological malignancy.

Levels of active tyrosine kinase receptor determine the tumor response to Zalypsis.

BACKGROUND: Zalypsis(®) is a marine compound in phase II clinical trials for multiple myeloma, cervical and endometrial cancer, and Ewing's sarcoma. However, the determinants of the response to Zalypsis are not well known. The identification of biomarkers for Zalypsis activity would also contribute to broaden the spectrum of tumors by selecting those patients more likely to respond to this therapy. METHODS: Using in vitro drug sensitivity data coupled with a set of molecular data from a panel of sarcoma cell lines, we developed molecular signatures that predict sensitivity to Zalypsis. We verified these results in culture and in vivo xenograft studies. RESULTS: Zalypsis resistance was dependent on the expression levels of PDGFRα or constitutive phosphorylation of c-Kit, indicating that the activation of tyrosine kinase receptors (TKRs) may determine resistance to Zalypsis. To validate our observation, we measured the levels of total and active (phosphorylated) forms of the RTKs PDGFRα/ß, c-Kit, and EGFR in a new panel of diverse solid tumor cell lines and found that the IC50 to the drug correlated with RTK activation in this new panel. We further tested our predictions about Zalypsis determinants for response in vivo in xenograft models. All cells lines expressing low levels of RTK signaling were sensitive to Zalypsis in vivo, whereas all cell lines except two with high levels of RTK signaling were resistant to the drug. CONCLUSIONS: RTK activation might provide important signals to overcome the cytotoxicity of Zalypsis and should be taken into consideration in current and future clinical trials.

PDGF receptor alpha+ mesoderm contributes to endothelial and hematopoietic cells in mice.

BACKGROUND: Early mesoderm can be classified into Flk-1+ or PDGF receptor alpha (PDGFRα)+ population, grossly representing lateral and paraxial mesoderm, respectively. It has been demonstrated that all endothelial (EC) and hematopoietic (HPC) cells are derived from Flk-1+ cells. Although PDGFRα+ cells give rise to ECs/HPCs in in vitro ES differentiation, whether PDGFRα+ population can become hemato-endothelial lineages has not been proved in mouse embryos. RESULTS: Using PDGFRαMerCreMer mice, PDGFRα+ early mesoderm was shown to contribute to endothelial cells including hemogenic ECs, fetal liver B lymphocytes, and Lin-Kit+Sca-1+ (KSL) cells. Contribution of PDGFRα+ mesoderm into ECs and HPCs was limited until E8.5, indicating that PDGFRα+/Flk-1+ population that exists until E8.5 may be the source for hemato-endothelial lineages from PDGFRα+ population. The functional significance of PDGFRα+ mesoderm in vascular development and hematopoiesis was confirmed by genetic deletion of Etv2 or restoration of Runx1 in PDGFRα+ cells. Etv2 deletion and Runx1 restoration in PDGFRα+ cells resulted in abnormal vascular remodeling and rescue of fetal liver CD45+ and Lin-Kit+Sca-1+ (KSL) cells, respectively. CONCLUSIONS: Endothelial and hematopoietic cells can be derived from PDGFRα+ early mesoderm in mice. PDGFRα+ mesoderm is functionally significant in vascular development and hematopoiesis from phenotype analysis of genetically modified embryos.

Expression of cell surface markers during self-renewal and differentiation of human adipose-derived stem cells.

Human adipose-derived stem cell populations express cell surface markers such as CD105, CD73, CD146 and CD140a/PDFGRα. However, it was unclear whether these markers could discriminate subpopulations of undifferentiated cells and whether the expression of these markers is modulated during differentiation. To address this issue, we analysed the immunophenotype of cultured human multipotent adipose derived stem (hMADS) cell populations at different adipocyte differentiation steps. We found that 100% of undifferentiated cells expressed CD73 and CD105. In contrast, CD146 and CD140a/PDFGRα marked two different subpopulations of cells. CD140a/PDGFRα subpopulation was regulated by FGF2, a critical factor of human adipose-derived stem cell self-renewal. During differentiation, CD73 was maintained and marked lipid-laden cells, whereas CD105 expression was inhibited in fully differentiated cells. The percentage of CD146 and CD140a/PDFGRα-positive cells declined as soon as cells had undergone differentiation. Altogether, these data support the notion that expanded adipose-derived stem cells are heterogeneous mixtures of cells and cell surface markers studied can discriminate subpopulations.

Specific analysis of KIT and PDGFR-alpha expression and mutational status in Merkel cell carcinoma.

BACKGROUND: The purpose of this study was to explore the immunohistochemical and mutational status of the tyrosine kinases KIT and platelet derived growth receptor-alpha (PDGFRA) in Merkel cell carcinoma (MCC). Specifically, we examined the mutated exons in gastrointestinal stromal cell tumors that may confer a treatment response to imatinib mesylate. METHODS: We evaluated KIT and PDGFRA immunostaining in 23 examples of MCC utilizing laser capture microdissection to obtain pure samples of tumor genomic DNA from 18 of 23 examples of MCC. PCR amplification and sequencing of KIT exons 9, 11, 13 and 17, and PDGFRA exons 10, 12, 14 and 18 for mutations was performed. RESULTS: Fifteen of 23 tumors (65%) demonstrated CD117 expression and 22 of 23 tumors (95%) demonstrated PDGFRA expression. A single heterozygous KIT exon 11 base change resulting in an E583K mutation was discovered in 12 of 18 (66%) examples of MCC. In addition, a single nucleotide polymorphism was detected in eight of 18 tumors (44%) in exon 18 of PDGFRA (codon 824; GTC > GTT). CONCLUSIONS: We discovered a novel somatic KIT exon 11 E583K mutation in 66% of tumors. This mutation has been previously described in a human with piebaldism and appears to represent an inactivating mutation. Therefore, despite expression of CD117 and PDGFRA, the absence of activating mutations in these tyrosine kinases makes KIT and PDGFRA unlikely candidates of MCC oncogenesis.

Repression of PDGF-R-α after cellular injury involves TNF-α, formation of a c-Fos-YY1 complex, and negative regulation by HDAC.

Wound healing is a complex dynamic process involving a variety of cell types, including fibroblasts that express and respond to cytokines and growth factors in the local microenvironment. The mechanisms controlling gene expression after injury at a transcriptional level are poorly understood. Here we show that decreased expression of a key receptor, PDGF-receptor (R)-α, after fibroblast injury is due to the release and paracrine activity of TNF-α. TNF-α inhibits PDGF-R-α expression and this involves formation of a c-Fos-Yin Yang 1 (YY1) complex and histone deacetylase (HDAC) activity. c-Fos, induced by TNF-α, negatively regulates PDGF-R-α transcription. Small interfering RNA (siRNA) targeting c-Fos or the zinc finger transcription factor YY1 inhibits TNF-α suppression of PDGF-R-α expression. Coimmunoprecipitation studies show that TNF-α stimulates the formation of a complex between c-Fos with YY1. Furthermore, chromatin immunoprecipitation (ChIP) analysis reveals the enrichment of c-Fos, YY1, and HDAC-1 at the PDGF-R-α promoter in cells exposed to TNF-α. With suberoylanilide hydroxamic acid (SAHA) and HDAC-1 siRNA, we demonstrate that HDAC mediates TNF-α repression of PDGF-R-α. These findings demonstrate that transcriptional repression of PDGF-R-α after fibroblast injury involves paracrine activity of endogenous TNF-α, the formation of a c-Fos-YY1 complex, and negative regulatory activity by HDAC.

The Dkk1 dose is critical for eye development.

During mammalian ocular development, several signaling pathways control the spatiotemporal highly defined realization of the three-dimensional eye architecture. Given the complexity of these inductive signals, the developing eye is a sensitive organ for several diseases. In this study, we investigated a Dkk1+/- haploinsufficiency during eye development, resulting in coloboma and anterior eye defects, two common developmental eye disorders. Dkk1 impacts eye development from a defined developmental time point on, and is critical for lens separation from the surface ectoderm via ß-catenin mediated Pdgfrα and E-cadherin expression. Dkk1 does not impact the dorso ventral retina patterning in general but is critical for Shh dependent Pax2 extension into the midline region. The described results also indicate that the retinal Dkk1 dose is critical for important steps during eye development, such as optic fissure closure and cornea formation. Further analysis of the relationship between Dkk1 and Shh signaling revealed that Dkk1 and Shh coordinatively control anterior head formation and eye induction. During eye development itself, retinal Dkk1 activation is depending on cilia mediated Gli3 regulation. Therefore, our data essentially improve the knowledge of coloboma and anterior eye defects, which are common human eye developmental defects.

The cellular prion protein identifies bipotential cardiomyogenic progenitors.

RATIONALE: The paucity of specific surface markers for cardiomyocytes and their progenitors has impeded the development of embryonic or pluripotent stem cell-based transplantation therapy. Identification of relevant surface markers may also enhance our understanding of the mechanisms underlying differentiation. OBJECTIVE: Here, we show that cellular prion protein (PrP) serves as an effective surface marker for isolating nascent cardiomyocytes as well as cardiomyogenic progenitors. METHODS AND RESULTS: Embryonic stem (or embryo-derived) cells were analyzed using flow cytometry to detect surface expression of PrP and intracellular myosin heavy chain (Myhc) proteins. Sorted cells were then analyzed for their differentiation potential. CONCLUSIONS: PrP+ cells from beating embryoid bodies (EBs) frequently included nascent Myhc+ cardiomyocytes. Cultured PrP+ cells further differentiated, giving rise to cardiac troponin I+ definitive cardiomyocytes with either an atrial or a ventricular identity. These cells were electrophysiologically functional and able to survive in vivo after transplantation. Combining PrP with a second marker, platelet-derived growth factor receptor (PDGFR)alpha, enabled us to identify an earlier cardiomyogenic population from prebeating EBs, the PrP+PDGFRalpha+ (PRa) cells. The Myhc- PRa cells expressed cardiac transcription factors, such as Nkx2.5, T-box transcription factor 5, and Isl1 (islet LIM homeobox 1), although they were not completely committed. In mouse embryos, PRa cells in cardiac crescent at the 1 to 2 somite stage were Myhc+, whereas they were Myhc- at headfold stages. PRa cells clonally expanded in methlycellulose cultures. Furthermore, single Myhc- PRa cell-derived colonies contained both cardiac and smooth muscle cells. Thus, PrP demarcates a population of bipotential cardiomyogenic progenitor cells that can differentiate into cardiac or smooth muscle cells.

The lack of CD34 expression in gastrointestinal stromal tumors is related to cystic degeneration following imatinib use.

OBJECTIVE: We evaluated the characteristics of the gastrointestinal stromal tumors that showed discrepancies between their assessment using the Response Evaluation Criteria in Solid Tumor (RECIST) and Choi's criteria. We also investigated the clinical applicability of Choi's criteria to Korean gastrointestinal stromal tumor patients undergoing imatinib therapy. METHODS: Patients with advanced gastrointestinal stromal tumors treated with frontline imatinib were analyzed. Computed tomography images of these patients were reviewed and genotyping for the KIT and PDGFRA genes was performed. Immunohistochemical staining of c-KIT, CD34, platelet derived growth factor receptor-alpha, platelet derived growth factor receptor-beta, AKT, P-ERK and vascular endothelial growth factor was followed. RESULTS: Ninety-five patients were enrolled. When using Choi's criteria to evaluate the 61 patients who achieved at least partial response by Choi's criteria, 27 patients showed discrepancies in their response to treatment between these two sets of criteria. A lack of CD34 expression in tumors was found to be related to cystic degeneration after imatinib treatment (P=0.001). Patients who showed partial response by Choi's criteria but stable disease by RECIST criteria had a similar progression-free survival to cases who showed a partial response under both systems (P=0.951). CONCLUSIONS: Gastrointestinal stromal tumors showing cystic degeneration after imatinib treatment lack CD34 expression. Choi's criteria have a clinical value in terms of the progression-free survival in Korean patients treated with imatinib.

Preclinical testing of tandutinib in a transgenic medulloblastoma mouse model.

Overexpression of platelet-derived growth factor receptor alpha (PDGFR-A) has been documented in association with primary tumors and metastasis in medulloblastoma. Tumors from our genetically engineered sonic hedgehog-driven medulloblastoma mouse model overexpress PDGFR-A in primary tumors and thus this mouse model is a good platform with which to study the role of PDGFR-A in this central nervous system malignancy. We hypothesized that inhibition of PDGFR-A in medulloblastoma can slow or inhibit tumor progression in living individuals. To test our hypothesis, we targeted PDGFR-A mediated tumor growth in vitro and in vivo using the tyrosine kinase inhibitor, tandutinib (MLN-518), which strongly inhibits PDGFR-A. Although PDGFR-A inhibition by this agent resulted in reduced mouse tumor cell growth and increased apoptosis in vitro, and reduced tumor cell proliferation in vivo, tandutinib did reduce tumor volume at the doses tested (360 mg/kg) in vivo. Thus, tandutinib may be an agent of interest for sonic hedgehog-driven medulloblastoma if a synergistic drug combination can be identified.

Transcriptome analysis reveals dialogues between human trophectoderm and endometrial cells during the implantation period.

BACKGROUND: Crosstalk between human trophectoderm (TE) and endometrial cells during the implantation window is a complex and not well-understood process. The aims of this study were (i) to evaluate the global gene expression profile in TE cells from Day 5 human blastocysts issued from IVF, (ii) to compare these data with the transcriptomic profile of endometrial cells in stimulated cycles for IVF and (iii) to identify potential early dialogues between maternal and embryonic cells during the implantation window. METHODS: Endometrial biopsies (n = 18) from normal responder patients were performed on the day of embryo transfer (Day 5 after human chorionic gonadotrophin administration). TE biopsies from five blastocysts donated for research purposes were mechanically extracted. DNA microarray analysis was carried out to identify the specific gene expression profiles and the biological pathways activated during the implantation window in endometrial and TE cells. RESULTS: Several cytokines (such as PDGFA, placenta growth factor, IGF2BP1 and IGF2BP3) were up-regulated in human TE cells, whereas some of the corresponding receptors (PDGFRA and KDR) were over-expressed in the receptive endometrium, suggesting that these molecules are involved in the early dialogue between blastocyst and maternal endometrial cells. In addition, several adhesion molecules and extracellular matrix proteins (MCAM, ITGAE and LAMA1) were also over-expressed in the TE, while others (ALCAM, CEACAM1, PECAM1, ITGB8 and LAMA2) were restricted to the receptive endometrium. CONCLUSION: The present study shows that several growth factors, cytokines, integrins and adhesion molecules are expressed in the TE and endometrium at the time of implantation. These results could contribute to the understanding of the mechanisms involved in the early dialogue between blastocyst and endometrium during implantation. Such results should be confirmed by further studies.

Expression of angiogenic basic fibroblast growth factor, platelet derived growth factor, thrombospondin-1 and their receptors at the porcine maternal-fetal interface.

BACKGROUND: Commercial swine breeds in North America undergo two waves of spontaneous fetal loss; one during peri-attachment and another during mid-gestation. Although an exact mechanism for this loss is not known, deficits in vasculature at the attachment sites appear to be a major cause. We hypothesized that a balance between pro-angiogenic and anti-angiogenic factors is needed at the maternal-fetal interface for successful conceptus development. Six selected members of the pro-angiogenic fibroblast growth factor (FGF) and platelet derived growth factor (PDGF) families and anti-angiogenic factor thrombospondin-1 (TSP-1) and its receptor CD36 were quantified and localized at the porcine maternal-fetal interface at early and midgestation time points. METHODS: Mesometrial endometrium was collected from non-pregnant gilts (n = 8). Endometrial and chorioallantoic membrane samples were collected from healthy and arresting conceptus attachment sites at gestation day (gd) 20 (n = 8) and gd 50 (n = 8). At gd20 arresting conceptus attachment sites were distinguished by decreased vasculature of the placental membranes and decreased conceptus size. At gd50 arresting conceptuses attachment sites were identified by smaller conceptus length and weight measurements. Quantitative real time PCR was used to determine relative transcript levels of genes of interest, and cellular localization was determined by immunohistochemistry in paraffin embedded endometrial sections. RESULTS: At gd20, endometrial samples from arresting conceptuses had elevated transcripts for bFGF, and PDGF-bb than healthy sites (p < 0.05). At gd50, bFGF, FGFR2, and CD36 were more abundant at arresting than at healthy conceptus attachment sites (p < 0.05). Chorioallantoic membrane from arresting conceptus attachment sites at gd20 had elevated transcripts for bFGF, FGFR1, FGFR2 and CD36 compared with healthy sites (p < 0.05). FGFR2 transcripts were more abundant in chorioallantoic membrane from arresting conceptuses at gd 50 (p < 0.05). Immunohistochemical localization of selected pro- and anti-angiogenic factors and receptors revealed their abundance in the luminal epithelium, uterine glands and perivascular areas of endometrium at gd20 and gd50. CONCLUSIONS: We provide comprehensive analysis of pro and anti-angiogenic factors at the porcine maternal fetal interface during early and mid-pregnancy. At mRNA levels, the majority of pro-angiogenic factors investigated were elevated at the sites of fetal arrest. These observations contrast with our previous findings of decreased Vascular Endothelial Growth Factor (VEGF) family members at arresting sites, and suggest that the bFGF family functions as a compensatory survival mechanism when major angiogenic proteins are decreasing at the sites of fetal arrest.

Hypomyelination phenotype caused by impaired differentiation of oligodendrocytes in Emx1-cre mediated Cdk5 conditional knockout mice.

Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in neuronal migration and differentiation, and in axonal elongation. Although many studies have been conducted to analyze neuronal functions of Cdk5, its kinase activity has also been reported during oligodendrocyte differentiation, which suggests Cdk5 may play an important role in oligodendrocytes. Here, we describe a hypomyelination phenotype observed in Emx1-cre mediated Cdk5 conditional knockout (cKO) mice (Emx1-cKO), in which the Cdk5 gene was deleted in neurons, astrocytes and oligodendrocyte -lineage cells. In contrast, the Cdk5 gene in CaMKII cKO mice was deleted only in neurons. Because the development of mature oligodendrocytes from oligodendrocyte precursor cells is a complex process, we performed in situ hybridization using markers for the oligodendrocyte precursor cell and for the differentiated oligodendrocyte. Our results indicate that hypomyelination in Emx1-cKO is due to the impaired differentiation of oligodendrocytes, rather than to the proliferation or migration of their precursors. The present study confirmed the in vivo role of Cdk5 in oligodendrocyte differentiation.

A Comparative Assessment of Marker Expression Between Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells and the Developing Pig Heart.

The course of differentiation of pluripotent stem cells into cardiomyocytes and the intermediate cell types are characterized using molecular markers for different stages of development. These markers have been selected primarily from studies in the mouse and from a limited number of human studies. However, it is not clear how well mouse cardiogenesis compares with human cardiogenesis at the molecular level. We tackle this issue by analyzing and comparing the expression of common cardiomyogenesis markers [platelet-derived growth factor receptor, alpha polypeptide (PDGFR-α), fetal liver kinase 1 (FLK1), ISL1, NK2 homeobox 5 (NKX2.5), cardiac troponin T (CTNT), connexin43 (CX43), and myosin heavy chain 7 (MYHC-B)] in the developing pig heart at embryonic day (E)15, E16, E18, E20, E22, and E24 and in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSCs). We found that porcine expression of the mesoderm marker FLK1 and the cardiac progenitor marker ISL1 was in line with our differentiating hiPSC and reported murine expression. The cardiac lineage marker NKX2.5 was expressed at almost all stages in the pig and hiPSC, with an earlier onset in the hiPSC compared with reported murine expression. Markers of immature cardiomyocytes, CTNT, and MYHC-B were consistently expressed throughout E16-E70 in the pig, which is comparable with mouse development, whereas the markers increased over time in the hiPSC. However, the commonly used mature cardiomyocyte marker, CX43, should be used with caution, as it was also expressed in the pig mesoderm, as well as hiPSC immature cardiomyocytes, while this has not been reported in mice. Based on our observations in the various species, we suggest to use FLK1/PDGFR-α for identifying cardiac mesoderm and ISL1/NKX2.5 for cardiac progenitors. Furthermore, a combination of two or more of the following, CTNT+/MYHC-B+/ISL1+ could mark immature cardiomyocytes and CTNT+/ISL1- mature cardiomyocytes. CX43 should be used together with sarcomeric proteins. This knowledge may help improving differentiation of hiPSC into more in vivo-like cardiac tissue in the future.