Effects of hydrogel three-dimensional culture on the characteristics of human amniotic mesenchymal stem cells and paracrine effect / 中国组织工程研究

BACKGROUND: Studies have shown that mesenchymal stem cells can reduce inflammation, promote wound healing, and reduce scar formation in wound healing. However, previous two-dimensional culture environment can lead to differences in gene expression, signal transduction, and morphology because of intracellular contact inhibition. OBJECTIVE: To investigate whether the ability of wound healing related factors secreted by human amniotic mesenchymal stem cells is affected by two-dimensional or three-dimensional culture environment. METHODS: The human amniotic mesenchymal stem cells cultured by traditional enzyme digestion method were inoculated in traditional cell culture flask (two-dimensional culture group) and ShakeGelTM 3D hydrogel (three-dimensional culture group) and induced to differentiate into adipocytes, osteoblasts, and chondrocytes, respectively. The direction of cell differentiation was determined by immunofluorescence staining. Human amniotic mesenchymal stem cells were fused to 70%-80% in two culture environments, and the growth characteristics and morphology of cells were observed under inverted phase contrast microscope and laser confocal microscope. After 24 hours of culture, relative mRNA expression of wound healing-related factors was detected by reverse transcription-quantitative polymerase chain reaction. After 48 hours of culture, the protein expression of wound healing-related factors was detected by the enzyme-linked immunosorbent assay. RESULTS AND CONCLUSION: (1) Human amniotic mesenchymal stem cells cultured in two-dimensional culture group were flat and spindle-shaped, which was a typical mesenchymal stem cell-like morphology. Human amniotic mesenchymal stem cells in the three-dimensional culture group were round and evenly dispersed in each layer of the hydrogel. (2) Human amniotic mesenchymal stem cells in the three-dimensional culture group exhibited the potential to differentiate into adipocytes, osteoblasts, and chondrocytes. (3) The mRNA expression of interleukin-6, interleukin-8, epidermal growth factor, basic fibroblast growth factor, hyaluronic acid, hepatocyte growth factor, and vascular endothelial growth factor in the three-dimensional culture group was significantly higher than that in the two-dimensional culture group (P 0.05). (4) The protein expression of interleukin-6, interleukin-8, interleukin-10, epidermal growth factor, basic fibroblast growth factor, hepatocyte growth factor, transforming growth factor β1 and vascular endothelial growth factor in the three-dimensional culture group was significantly higher than that in the two-dimensional culture (P 0.05). (5) These findings suggest that human amniotic mesenchymal stem cells cultured in three-dimensional hydrogel show better morphology and more encouraging paracrine effect of wound healing related factors than those cultured in traditional two-dimensional culture environment.

Biological characteristics of endometrial stem cells isolated from ectopic lesions of endometriosis / 解剖学报

Objective: To establish the approach of isolating, culturing and identifing endometrial stem cells (EnSCs) derived from ectopic lesion of endometriosis patient; and preliminarily examine the biological characteristic of ectopic EnSCs, which provide support for further study on the potential role of ectopic EnSCs in the pathogenesis of endometriosis. Methods: The ectopic lesions of endometriosis were harvested from the patients with the informed consent and transferred to lab as soon as possible. The ectopic lesions were minced, digested by collagenase I and seeded into cell culture flasks for conventional culture (n=10). Expression of vimentin in ectopic EnSCs was examined by immunofluorescence (n = 3). Proliferative capacity of ectopic EnSCs was examined by MTT assay (n = 5). Multilineage differentiation potential of ectopic EnSCs was examined by adipogenic and osteogenic differentiation respectively (n = 3). Immunophenotype analysis of ectopic EnSCs was determined by flow cytometry (n = 3). Production of biological factors in ectopic EnSCs derived conditional medium (n = 6) and expression of adhesion molecules on ectopic EnSCs (n = 7) were examined by protein assays. Results: We successfully isolated EnSCs from ectopic lesions of endometriosis patients, and ectopic EnSCs were positive for vimentin and typical markers of mesenchymal stem cell (CD29, CD73, CD90 and CD105), and negative for the markers of hematopoietic stem cell (CD34 and CD45). The induced ectopic EnSCs showed obvious lipid droplets (adipogenic differentiation) and calcium nodules (osteogenic differentiation). The ectopic EnSCs could secrete high concentration of angiogenic factors [vascular endothelial growth factor (VEGF), angiotensin (ANG) and platelet-derived growth factor (PDGF)-AA]and angiogenesis associated inflammation cytokines [interleukin (IL-6), IL-8 and monocyte chemotactic protein l(MCP-l)]. Additionally, adhesion molecules analysis demonstrated the high expression of activated leukocyte adhesion molecule (ACLAM) and intercellular cell adhesion molecule-1 (ICAM-1) on ectopic EnSCs. Conclusion: We successfully establish the procedure of isolating and culturing ectopic EnSCs and demonstrate that ectopic EnSCs is capable of promoting angiogenesis through secreting high concentration of associated biological factors. The above result confirm the existence of EnSCs in ectopic lesions of endometriosis, which not only supports the stem cell based pathogenesis of endometriosis, but also shows the therapeutic potential of taking ectopic EnSCs as promising targets in the treatment of endometriosis.

Research progress in treatment of knee osteoarthritis by paracrine effect of stem cells / 中国修复重建外科杂志

Objective: To review the advances in utilizing paracrine effect of stem cells in knee osteoarthritis (OA) treatment. Methods: The researches in applying stem cells derived conditioned medium, extracellular matrix, exosomes, and microvesicles in knee OA treatment and cartilage repair were reviewed and analyzed. Results: The satisfying outcomes of using different products of stem cells paracrine effect in knee OA condition as well as cartilage defect is revealed in studies in vitro and in vivo. The mechanism including suppressing the intraarticular inflammation, the apoptosis of chondrocytes, and the degradation of cartilage matrix, while enhancing the synthesis of cartilage matrix, the differentiation of in-situ stem cells into chondrocytes and the migration to the affected area. The effectiveness can be further improved supplemented with the tissue engineering methods or gene modification. Conclusion: Compared with the traditional stem cell therapy, applying the products from paracrine effect of stem cells in knee OA treatment is more economical and safer, presenting great potential in clinical practice.

Paracrine influence of human perivascular cells on the proliferation of adenocarcinoma alveolar epithelial cells

Understanding the crosstalk mechanisms between perivascular cells (PVCs) and cancer cells might be beneficial in preventing cancer development and metastasis. In this study, we investigated the paracrine influence of PVCs derived from human umbilical cords on the proliferation of lung adenocarcinoma epithelial cells (A549) and erythroleukemia cells (TF-1α and K562) in vitro using Transwell® co-culture systems. PVCs promoted the proliferation of A549 cells without inducing morphological changes, but had no effect on the proliferation of TF-1α and K562 cells. To identify the factors secreted from PVCs, conditioned media harvested from PVC cultures were analyzed by antibody arrays. We identified a set of cytokines, including persephin (PSPN), a neurotrophic factor, and a key regulator of oral squamous cell carcinoma progression. Supplementation with PSPN significantly increased the proliferation of A549 cells. These results suggested that PVCs produced a differential effect on the proliferation of cancer cells in a cell-type dependent manner. Further, secretome analyses of PVCs and the elucidation of the molecular mechanisms could facilitate the discovery of therapeutic target(s) for lung cancer.

Liver Regenerating Potential of the Secretome Obtained from Adipose-derived Stem Cells Cultured under the Hypoxic Environment / 대한이식학회지

BACKGROUND: The stem cell-derived secretome has received considerable attention as an alternative to stem cells for therapeutic applications. However, establishing optimal culture conditions is key to obtaining appropriate secretome contents. Here, the optimal culturing environment for achieving a high-efficiency secretome was determined via hypoxic preconditioning of human adipose-derived stem cells (ASC). METHODS: Normoxic conditioned media (NCM) and hypoxic conditioned media (HCM) were obtained after culturing human ASCs under normoxia (20% O2) or hypoxia (1% O2), respectively. Subsequently, both normal and thioacetamide-induced hepatotoxic hepatocytes were treated with NCM or HCM. In addition, partially hepatectomized mice were infused with control saline, NCM, and HCM. The effects on liver regeneration and serum transaminases levels were then compared. RESULTS: Hypoxic preconditioning significantly increased mRNA expression of proinflammatory cytokines (interleukin-6 and tumor necrosis factor-α) and growth factors (hepatocyte growth factor and vascular endothelial growth factor). In both normal and thioacetamide-induced hepatotoxic hepatocyte (alpha mouse liver 12 [AML12]) cell lines, HCM treatment resulted in the highest cell viability (122% and 95%, respectively), followed by NCM (111% and 78%, respectively). In addition, intravenous administration of HCM to partially hepatectomized mice resulted in substantially enhanced liver regeneration compared with the NCM group (P<0.05). CONCLUSIONS: Taken together, the secretome obtained from ASC with hypoxic preconditioning showed potential to alleviate liver damage both in vitro and in vivo. Hypoxic culture of ASC is expected to play an important role in regenerative medicine by inducing secretome production that is beneficial for improving liver regeneration.

Paracrine research progress of bone marrow mesenchymal stem cells / 中国小儿急救医学

In recent years,along with the deepening study of bone marrow mesenchymal stem cells (BMSCs),the repair effect of BMSCs in various tissue injury have been gradually revealed.And in recent years,except the ability of differentiate to the target cells,its paracrine effect,for example,a variety of cyto-kines secreted by BMSCs,also plays an important role in the process of repairing.

CPC derivedexosome protects cardiomyocytes from oxidative stress / 中国药理学通报

Aim To explore the anti-apoptotic function of cardiac progenitor cells(CPCs)-derived exosome in vitro.Method CPCs were isolated from mouse heart using Magnetic Cell Sorting(MACS)system.Flow Cy-tometry(FC)determine the purity of stem cell surface antigen-1 positive(Sca-1 +)CPCs.Exosome was puri-fied from conditional medium,and confirmed by West-ern blot using CD63 as a marker,Nanoparticle Traffic-king Analysis(NTA)was used to detect the diameters and concentration of exosome.Then the cells were di-vided into control groups and CPC-exosome pre-protec-tion groups.H2 O2 was added into H9c2 cells to induce oxidative stress.Western blot was adopted to determine the expression of cleaved caspase-3.Results ① Im-munofluorescence showed that CPCs isolated by MACS were positively expressing Sca-1 protein;FC analysis showed that typical purity of Sca-1 +CPCs from the first preparations was more than 95%.② WB demonstrated that CD63 of exosome isolated from CCMwas positively expressed,and NTA results showed that the diameters of exosome were (82.33 ±3.06)nm(n =3).Micro-scope detected PKH-26 labeled exosome appeared in the cytoplasma of H9c2 cells.③ Western blot showed the CPC-exosome pre-protection groups significantly down-regulated the levels of cleaved caspase-3 com-pared to the control groups(P <0.05).Conclusion CPC can secrete exosome which carries many important cargos,which can effectively gather in H9c2 cells. CPC-exosome can protect H9c2 cells from the oxidative stress induced by H2 O2 .Our results highlight a new perspective strategy for cardiac disease.

Neurorestoration Induced by Mesenchymal Stem Cells: Potential Therapeutic Mechanisms for Clinical Trials

Stem cells are emerging as therapeutic candidates in a variety of diseases because of their multipotent capacities. Among these, mesenchymal stem cells (MSCs) derived from bone marrow, umbilical cord blood or adipose tissue, comprise a population of cells that exhibit extensive proliferative potential and retain the ability to differentiate into multiple tissue-specific lineage cells including osteoblasts, chondrocytes, and adipocytes. MSCs have also been shown to enhance neurological recovery, although the therapeutic effects seem to be derived from an indirect paracrine effect rather than direct cell replacement. MSCs secrete neurotrophic factors, promote endogenous neurogenesis and angiogenesis, encourage synaptic connection and remyelination of damaged axons, decrease apoptosis, and regulate inflammation primarily through paracrine actions. Accordingly, MSCs may prevail as a promising cell source for cell-based therapy in neurological diseases.

Medicina regenerativa y células madre. Mecanismos de acción de las células madre adultas / Regenerating medicine related to the stem-cells and its mechanisms of action from adults cells

La medicina regenerativa es una rama de la medicina que se ha desarrollado considerablemente en los últimos años. Los avances en este campo se han vinculado estrechamente con los nuevos conocimientos adquiridos sobre las células madre y su capacidad de convertirse en células de diferentes tejidos. Esta medicina se sustenta en conductas adoptadas por el organismo para remplazar por células sanas a las dañadas por diversos procesos en determinados tejidos. Las medidas terapéuticas empleadas pueden incluir trasplante de células madre, el uso de moléculas solubles, terapia génica e ingeniería de tejidos. En la actualidad, el método más empleado es el trasplante de células madre adultas. Sin embargo, todavía no se conocen bien los mecanismos mediante los cuales las células trasplantadas podrían mejorar o promover la regeneración de los tejidos. Para explicar estos mecanismos se han sugerido varias hipótesis, que incluyen la transdiferenciación celular, la fusión de células y los efectos secundarios a la liberación por las células de diferentes moléculas solubles con acciones específicas; además de los efectos autocrinos y paracrinos que pueden tener estos factores solubles se sugiere la existencia también de una acción telecrina. Probablemente se ejecute más de uno de estos mecanismos.

Regenerating medicine is a branch of Medicine very developed in past years. Advances in this field have been closely linked with the new knowledge achieved on stem-cells and its ability to become in cells of different tissues. This type of medicine is based on the behaviors adopted by organism to substitute those damaged cells by the healthy ones by different processes in specific tissues. Therapeutic measures used may include the stem-cell transplantation, the use of soluble molecules, genic therapy and tissues engineering. Nowadays, the more used method is the adult stem-cells. However, is not well known the mechanisms by which the transplanted cells could to improve or to promote the tissue regeneration. To explain these mechanisms some hypotheses has been proposed including the cellular trans-differentiation, cells fusion, and the effects secondaries to cells release by cells of different soluble molecules with specific actions; in addition to the autocrine and paracrine effects that may have these soluble factors, it is suggested too the existence of a telecrine action. It is probable that more than one of these mechanisms be executed.