Promoting Tissue Differentiation Research Articles

Hypoxia as a stimulus for tissue formation: The concept of organogenesis in microsurgically vascularized tissue engineering constructs

Axial vascularization of tissue constructs is essential to maintain an adequate blood supply for a stable regeneration of a clinically relevant tissue size. The versatility of the arterio-venous loop (AVL) has been previously shown in various small and large animal models as well as in clinical reports for bone regeneration. We have previously demonstrated the capability of the AVL to induce axial vascularization and to support the nourishment of tissue constructs in small animal models after applying high doses of ionizing radiation comparable to those applied for adjuvant radiotherapy after head and neck cancer. We hypothesize that this robust ability to induce regeneration after irradiation could be related to a state of hypoxia inside the constructs that triggers the HIF1 (hypoxia induced factor 1) - SDF1 (stromal derived factor 1) axis leading to chemotaxis of progenitor cells and induction of tissue regeneration and vascularization. We analyzed the expression of HIF1 and SDF1 via immunofluorescence in axially vascularized bone tissue engineering constructs in Lewis rats 2 and 5 weeks after local irradiation with 9Gy or 15Gy. We also analyzed the expression of various genes for osteogenic differentiation (collagen 1, RUNX, alkaline phosphatase and osteonectin) via real time PCR analysis. The expression of HIF1 and SDF1 was enhanced two weeks after irradiation with 15Gy in comparison to non-irradiated constructs. The expression of osteogenic markers was enhanced at the 5-weeks time point with significant results regarding collagen, alkaline phosphatase and osteonectin. These results indicate that the hypoxia within the AVL constructs together with an enhanced SDF1 expression probably play a role in promoting tissue differentiation. The process of tissue generation triggered by hypoxia in the vicinity of a definite vascular axis with enhanced tissue differentiation over time resembles hereby the well-known concept of organogenesis in fetal life.

Easy-to-Build and Reusable Microfluidic Device for the Dynamic Culture of Human Bronchial Cystic Fibrosis Epithelia.

Cystic fibrosis (CF) is one of the most frequent genetic diseases, caused by dysfunction of the CF transmembrane conductance regulator (CFTR) chloride channel. CF particularly affects the epithelium of the respiratory system. Therapies aim at rescuing CFTR defects in the epithelium, but CF genetic heterogeneity hinders the finding of a single and generally effective treatment. Therefore, in vitro models have been developed to study CF and guide patient therapy. Here, we show a CF model on-chip by coupling the feasibility of the human bronchial epithelium differentiated in vitro at the air-liquid interface and the innovation of microfluidics. We demonstrate that the dynamic flow enhanced cilia distribution and increased mucus quantity, thus promoting tissue differentiation in a short time. The microfluidic devices highlighted differences between CF and non-CF epithelia, as shown by electrophysiological measures, mucus quantity, viscosity, and the analysis of ciliary beat frequency. The described model on-chip may be a handy instrument for studying CF and setting up therapies. As a proof of principle, we administrated the corrector VX-809 on-chip and observed a decrease in mucus thickness and viscosity.

Chinese herbal medicine promote tissue differentiation in colorectal cancer by activating HSD11B2

BackgroundColorectal cancer is a common malignant tumor of the digestive tract. In recent years, the incidence rate has increased year by year and is showing a younger trend. The application of Chinese herbal medicine (CHM) is one of the important methods for the treatment of colorectal cancer. CHM refers to the main therapeutic drugs based on traditional Chinese medicine (TCM), which is still valued. Many effective anticancer small-molecule compounds are derived from CHMs, and their effective anticancer ingredients and targets must be clarified and to further understand the molecular mechanisms by which CHM affects cancer. MethodsWe analyzed the ingredients in CHM that were found to be effective against colorectal cancer and constructed an interaction network among these ingredients and the target protein. By analyzing the number of connections in the network and their type of interaction, we identified the key target protein Corticosteroid 11-beta-dehydrogenase isozyme 2, the enzyme encoded by HSD11B2. Analyses of HSD11B2 expression, survival curve, and co-expressed genes helped clarify the correlation between HSD11B2 and colorectal cancer as well as its underlying molecular mechanism. ResultsWe determined that the anticancer ingredients contained in Sanguisorba officinalis, Patrinia scabiosaefolia, and Smilax china had more connections to the target proteins found in colorectal cancer. In the interaction network, eight small-molecule compounds had an activating effect on HSD11B2. The expression of the HSD11B2 was markedly decreased in colorectal cancer tissues and was positively correlated with the overall survival time of patients. In addition, co-expression analyses showed a close relationship between HSD11B2 and tissue-specific genes in colorectal tissues. The expression levels of HSD11B2 in well-, moderately, and poorly differentiated tissues progressively decreased. ConclusionThe HSD11B2 protein was a key CHM target for treating colorectal cancer. The key role of CHM may lie in activating HSD11B2 and further promoting tissue differentiation in colorectal cancer.

Progress in the correlation between PTPN12 gene expression and human tumors.

Background:The global morbidity of cancer is rising rapidly. Despite advances in molecular biology, immunology, and cytotoxic and immune-anticancer therapies, cancer remains a major cause of death worldwide. Protein tyrosine phosphatase non-receptor type 12 (PTPN12) is a new member of the cytoplasmic protein tyrosine phosphatase family, isolated from a cDNA library of adult colon tissue. Thus far, no studies have reviewed the correlation between PTPN12 gene expression and human tumors.Methods:This article summarizes the latest domestic and international research developments on how the expression of PTPN12 relates to human tumors. The extensive search in Web of Science and PubMed with the keywords including PTPN12, tumor, renal cell carcinoma, proto-oncogenes, tumor suppressor genes was undertaken.Results:More and more studies have shown that a tumor is essentially a genetic disease, arising from a broken antagonistic function between proto-oncogenes and tumor suppressor genes. When their antagonistic effect is out of balance, it may cause uncontrolled growth of cells and lead to the occurrence of tumors. PTPN12 is a tumor suppressor gene, so inhibiting its activity will lead directly or indirectly to the occurrence of tumors.Conclusion:The etiology, prevention, and treatment of tumors have become the focus of research around the world. PTPN12 is a tumor suppressor gene. In the future, PTPN12 might serve as a novel molecular marker to benefit patients, and even the development of tumor suppressor gene activation agents can form a practical research direction.

Neuro-vascular Interactions during Neocortical Development-Systematic and Accurate Regulatory Mechanisms of VEGF Signaling

Blood vessels supply oxygen and nutrients to all the cells in a living body, and provide essential transport routes for collecting waste products. For these functions, blood vessel networks should be appropriately formed in each tissue. Therefore, blood vessels are one of the earliest organs formed during the developmental process. Development of the blood vessel system promotes tissue differentiation and organ morphogenesis, allowing each organ to maintain its unique functions under changing metabolic conditions. Blood vessels have a relatively simple structure, consisting of endothelial cells covering the inner layer, and pericytes or smooth muscle cells surrounding the outside. The structure of the vascular network is extremely diverse, with blood vessels uniquely organized depending on the tissues they serve, to create tissue-specific microenvironments. How are such tissue-specific vascular environments generated? Over the years, anatomical findings have accumulated to confirm this vascular diversity. However, the molecular basis for this diversity has remained unclear. In the present article, we review the mechanisms of coordinated developmental control of the vascular and neural systems in the cerebral cortex from the viewpoint of the accurate expression control of vascular endothelial growth factor (VEGF) signaling, and describe future perspectives.

Design strategies and applications of nacre-based biomaterials.

Mussel derived nacre, a biological composite composed of mineralized calcium carbonate platelets and interplatelet protein components, has recently gained interest as a potential alternative ceramic material in orthopedic biomaterials, combining the integration and mechanical capabilities of calcium phosphates with increased bioactivity derived from proteins and biomolecules; however, there is limited awareness of this material's potential. Herein, we present, to our knowledge, the first comprehensive review of nacre as a biomaterial. Nacre is a highly promising yet overlooked biomaterial for orthopedic tissue engineering with great potential in a wide variety of material systems. It is our hope that publication of this article will lead to increased community awareness of the potential of nacre as a versatile, bioactive ceramic capable of improving bone tissue regeneration and will elicit increased research effort and innovation utilizing nacre.

A molecular mechanism that confines the activity pattern of miR165 in Arabidopsis leaf primordia.

In Arabidopsis leaf primordia, the expression of HD-Zip III, which promotes tissue differentiation on the adaxial side of the leaf primordia, is repressed by miRNA165/166 (miR165/166). Small RNAs, including miRNAs, can move from cell to cell. In this study, HD-Zip III expression was strikingly repressed by miR165/166 in the epidermis and parenchyma cells on the abaxial side of the leaf primordia compared with those on the adaxial side. We also found that the MIR165A locus, which was expressed in the abaxial epidermis, was sufficient to establish the rigid repression pattern of HD-Zip III expression in the leaf primordia. Ectopic expression analyses of MIR165A showed that the abaxial-biased miR165 activity in the leaf primordia was formed neither by a polarized distribution of factors affecting miR165 activity nor by a physical boundary inhibiting the cell-to-cell movement of miRNA between the adaxial and abaxial sides. We revealed that cis-acting factors, including the promoter, backbone, and mature miRNA sequence of MIR165A, are necessary for the abaxial-biased activity of miR165 in the leaf primordia. We also found that the abaxial-determining genes YABBYs are trans-acting factors that are necessary for the miR165 activity pattern, resulting in the rigid determination of the adaxial-abaxial boundary in leaf primordia. Thus, we proposed a molecular mechanism in which the abaxial-biased patterning of miR165 activity is confined.

An In Vivo Evidence That the CD45negative Adult Marrow-Derived CXCR4+ SSEA-1+ OCT-4+ Very Small Embryonic-Like (VSEL) Stem Cells May Differentiate into CD45positive Long Term Repopulating Hematopoietic Stem Cells.

Mounting evidence accumulates that bone marrow (BM) contains a population of pluripotent stem cells (PSC) that give rise to long-term repopulating hematopoietic stem cells (HSC). Recently we identified in murine BM a homogenous population of rare (∼0.01% of BMMNC) and very small (2–4 μm) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of PSC such as SSEA-1, Oct-4, Nanog and Rex-1 and highly express Rif-1 telomerase protein (Leukemia 2006; 20,857–869). Direct electronmicroscopical analysis revealed that these cells display several features typical for primary epiblast-derived embryonic stem cells such as i) a large nuclei surrounded by a narrow rim of cytoplasm, and ii) open-type chromatin (euchromatin). In co-cultures with C2C12 murine sarcoma supportive feeder-layer, these cells grow spheres that are composed of immature CXCR4+SSEA-1+Oct-4+ cells that have large nuclei containing euchromatin and, if plated into cultures promoting tissue differentiation, show pluripotency and expand into cells from all three germ-cell layers. Based on this, we called them very small embryonic like (VSEL) stem cells. However, VSEL isolated freshly from the BM do not posses immediate hematopoietic activity - they neither grow hematopoietic colonies nor radioprotect lethally irradiated recipients. Recently, however, we noticed that if plated over supportive OP9 cell line, these CD45− VSEL give rise to colonies of CD45+CD41+Gr-1−Ter119− cells where their phenotype resembles those of the earliest hematopoietic cells that are derived in vitro from established embryonic cell lines. This hematopoietic differentiation of VSEL was accompanied by upregulation of mRNA for several genes regulating hematopoiesis (e.g. PU-1, c-myb, LMO2, Ikaros). More importantly, the CD45+CD41−Gr-1−Ter119− cells expanded from VSEL isolated from GFP+ mice if transplanted into wild-type animals protected them from lethal irradiation and differentiated in vivo into all major hematopoietic lineages (e.g., Gr-1+, B220+ and CD3+ cells). This hematopoietic activity was maintained after transplantation into secondary recipients. Based on this, we postulate that i) VSEL are PSC that give rise to HSC and ii) that CD45+ cells may derive from a CD45− population. Thus we propose that VSEL are a population of BM-residing PSC that may give rise to long-term engrafting hematopoietic stem cells.

Tie-dyed1Functions Non-Cell Autonomously to Control Carbohydrate Accumulation in Maize Leaves

The tie-dyed1 (tdy1) mutant of maize (Zea mays) produces chlorotic, anthocyanin-accumulating regions in leaves due to the hyperaccumulation of carbohydrates. Based on the nonclonal pattern, we propose that the accumulation of sucrose (Suc) or another sugar induces the tdy1 phenotype. The boundaries of regions expressing the tdy1 phenotype frequently occur at lateral veins. This suggests that lateral veins act to limit the expansion of tdy1 phenotypic regions by transporting Suc out of the tissue. Double mutant studies between tdy1 and chloroplast-impaired mutants demonstrate that functional chloroplasts are needed to generate the Suc that induces the tdy1 phenotype. However, we also found that albino cells can express the tdy1 phenotype and overaccumulate Suc imported from neighboring green tissues. To characterize the site and mode of action of Tdy1, we performed a clonal mosaic analysis. In the transverse dimension, we localized the function of Tdy1 to the innermost leaf layer. Additionally, we determined that if this layer lacks Tdy1, Suc can accumulate, move into adjacent genetically wild-type layers, and induce tdy1 phenotypic expression. In the lateral dimension, we observed that a tdy1 phenotypic region did not reach the mosaic sector boundary, suggesting that wild-type Tdy1 acts non-cell autonomously and exerts a short-range compensatory effect on neighboring mutant tissue. A model proposing that Tdy1 functions in the vasculature to sense high concentrations of sugar, up-regulate Suc transport into veins, and promote tissue differentiation and function is discussed.

The CD45−LIN− Adult Marrow-Derived CXCR4+ SSEA-1+ Oct-4+ Very Small Embryonic-Like (VSEL) Stem Cells Form In Vitro Spheres Which May Differentiate into CD45+ Hematopoietic Cells.

Recently we identified in murine BM a homogenous population of rare (~0.01% of BMMNC) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1 and highly express Rif-1 telomerase protein (Leukemia 2006; 20,857–869). Direct electronmicroscopical analysis revealed that these cells display several features typical for primary embryonic stem cells such as i) a small size (~2–4 μm in diameter), ii) a large nuclei surrounded by a narrow rim of cytoplasm, and iii) open-type chromatin (euchromatin). These cells isolated freshly from the BM neither grow hematopoietic colonies nor radioprotect lethally irradiated recipients. Recently, however, we noticed that purified VSELs in co-cultures with C2C12 murine sarcoma supportive feeder-layer grow spheres and cells from these VSEL-derived spheres (VSEL-DS) are composed of immature cells with large nuclei containing euchromatin, and similarly as VSELs are CXCR4+SSEA-1+Oct-4+. We found that cells from VSEL-DS after re-plating over C2C12 cells may again (up to 5–7 passages) grow new embryoid-like bodies or if plated into cultures promoting tissue differentiation show pluripotency and expand into cells from all three germ-cell layers. Based on this we tested if CD45− VSEL-DS cells could also differentiate into the hematopoietic lineage. To address this issue we employed similar culture conditions that are employed for hematopoietic differentiation of established embryonic stem cell lines. We noticed that VSEL-DS cells if cultured/passaged in methylocellulose cultures supplemented with hematopoietic growth factors (KL, IL-3, EpO and GM-CSF) give rise to colonies composed of myeloid (CD45+ Gr-1+) and erythroid (Terr-119+) hematopoietic cells. The hematopoietic differentiation of VSEL-DS cells was accompanied by upregulation of mRNA for several genes regulating hematopoiesis (e.g. PU-1, c-myb, LMO2, Ikaros). Based on this we postulate that VSELs that reside in bone marrow may contribute to hematopoiesis. We also provide direct evidence that CD45+ cells may derive from a CD45− population. We postulate that VSELs are closely related to a population of long-term engrafting hematopoietic stem cells and currently we are testing this possibility in animal models.

An Evidence That Murine Marrow-Derived CXCR4+ SSEA-1+ Oct-4+ Very Small Embryonic-Like (VSEL) Stem Cells Are Pluripotent and Express Several Primordial Germ Cell (PGC) Markers - Hypotesis for Developmental Deposition of PGC in Various Organs.

Recently we identified in murine BM a homogenous population of rare (~0.01% of BMMNC) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1 and highly express Rif-1 telomerase protein (Leukemia 2006; 20,857–869). Direct electronmicroscopical analysis revealed that these cells display several features typical for embryonic stem cells such asi) small size (2–4 um in diameter),ii) large nuclei surrounded by a narrow rim of cytoplasm, andiii) open-type chromatin (euchromatin).We also found that VSELs may be released from BM and circulate in peripheral blood during tissue/organ injuries (e.g., heart infarct, stroke). Recently we noticed that ~5–10% of purified VSELs if plated over a C2C12 murine sarcoma cell feeder layer are able to form spheres that resemble embryoid bodies. Cells from these VSEL-derived spheres (VSEL-DS) are composed of immature cells with large nuclei containing euchromatin, and similarly as purified VSELs are CXCR4+SSEA-1+Oct-4+. Furthermore, VSEL-DS after replating over C2C12 cells may again (up to 5–7 passages) grow new spheres or if plated into cultures promoting tissue differentiation expand into cells from all three germ-cell layers. The formation of VSEL-DS was observed in a presence of C2C12 cells obtained from different sources. Furthermore, VSELs isolated from GFP+ mice grew GFP+ VSEL-DS which show a diploid content of DNA. This suggests that VSEL-DS are in fact derived from VSELs and not from the supportive C2C12 cell line as well as excludes the possibility of cell fusion to the observed phenomenon. Similar spheres were also formed by VSELs isolated from murine fetal liver, spleen and thymus. Interestingly formation of VSEL-DS was associated with a young age, and no VSEL-DS were observed by cells isolated from old mice (> 2 years). We also found that cells isolated from VSEL-DS similarly as embryonic stem cells grow tumors after injection into immunodeficient NOD/SCID mice (51/52 inoculated mice). Since VSELs isolated by us express several markers of primordial germ cells (fetal-type alkaline phosphatase, Oct-4, SSEA-1, CXCR4, Mvh, Stella, Fragilis, Nobox, Hdac6) we hypothesize that VSELs are closely related to a population of primordial germ cells. These cells are specified during early gastrulation in the proximal epiblast and subsequently migrate in a CXCR4-SDF-1 dependent manner through the embryo proper to their final destination in genital ridges. It is possible that some of these cells or a population of cells closely related to them migrate astray being chemoattracted by SDF-1 to fetal liver and subsequently, during the third trimester of gestation seed together with hematopoietic stem cells in bone marrow and perhaps other organs as well. In conclusion, we postulate that VSELs identified by us and purified at the single cell level could become an important source of pluripotent stem cells for regeneration.

Creating permanent 3D arrangements of isolated cells using holographic optical tweezers

We report the creation of permanent 3D configurations of cells, at predefined positions, within a gelatin matrix. The technique used holographic optical tweezers to manipulate individual E. coli within a solution comprising monomer precursors. The matrix was then set and after the laser beam was removed, we were able to demonstrate that the structures remained intact for many days. We were also able to demonstrate that, in the presence of appropriate nutrients, the E. coli survived within the gelatin matrix for several days. The technique could have a number of potential future applications, including the arrangement of a variety of different cell types in complex architectures, as motifs for promoting tissue differentiation and growth within the field of cell engineering.

Cytological effects of hormones and plasma on bovine mammary tissue in vitro.

Mammary explants from two heifers pretreated with oestradiol and progesterone were cultured for 96 h with various combinations of hormones and calf plasma to identify the complement causing mammary tissue development in vitro in the cow. Initial tissue histology, determined by quantitative morphological analysis, was maintained by incubation with insulin, cortisol, oestrogen and progesterone; enlarged lumina were observed after treatment with insulin and cortisol. Lactogenesis was induced in vitro by insulin, cortisol and prolactin, enhanced by adding oestrogen and progesterone at the doses used here and further stimulated by the addition of plasma. The most highly developed mammary alveoli were characterized by an increased luminal area with lipid and stainable secretion, epithelia with large lipid droplets in the apical cytoplasm and a limited stromal area. In a second experiment, samples of plasma were collected from two cows, successfully induced to lactate, on days 6, 15, 17, 19 and 21 after treatment with oestradiol and progesterone. Mammary gland explants from five heifers pretreated with oestradiol and progesterone in vivo were cultured with insulin and cortisol plus these plasma samples (30%, v/v) to test for changes in lactogenic activity. All plasma samples were found equally beneficial in promoting tissue differentiation. These experiments show that low concentrations of ovarian steroids synergize with prolactin at the level of the mammary epitherlium and suggest that other plasma components aid the development of bovine mammary epithelium in vitro.

In vitro differentiation of tooth rudiments of embryonic mice. II. Growth of isolated thirds of embryonic mouse incisors

AbstractThe mandibular incisor of the 16 day embryonic mouse was divided into three visibly equal thirds; each third was explanted in vitro to test its capabilities for independent development. Ameloblasts and odontoblasts, present in the distal third at the time of isolation, fail to continue their differentiation. This third flattens, and the tissues become disorganized. Grouping several distal thirds in a single culture did not promote tissue differentiation. The embryonic proximal third remains cohesive The tissues differentiate; both ameloblasts and odontoblasts deposit extracellular matrix. Approximately half of the cultures of the intermediate third show differentiated odontoblasts and extracellular matrix after 11 culture days. Measurements of the volume changes of ameloblasts, odontoblasts, and general mesenchyme were taken over the first six culture days for each third of the rudiment. The volume changes recorded, and the counts of mitotic figures reflect the developmental behavior of each fraction of the rudiment.