Expression of mesenchymal stem cell phenotype in human nasal respiratory epithelial cells / Expresión del fenotipo de células troncales mesenquimales en células epiteliales nasales humanas

The respiratory epithelium is the first line of contact with the external hazards. Thus it can be damaged and need to be replaced to avoid healing by fibrosis. Tracheal tissue engineering is an alternative promising treatment modality. Mesenchymal stem cell markers are surface proteins, which are responsible for some of these cells unique properties. The objective of this study was to detect the mesenchymal stem cell phenotype among the human nasal respiratory epithelial cells via two immunophenotyping techniques. Respiratory epithelial cells were cultured using co-culture technique, fibroblasts was removed at confluence leaving respiratory epithelial cells, which were passage further to passage 4. Cells were evaluated for mesenchymal stem cell markers that were CD73, CD90, CD105 and the hematopoietic stem cell marker CD45 at passage 1 (P1) and passage 4 (P4) using Flow cytometry and Immunocytochemistry techniques. Respiratory epithelial cells expressed the mesenchymal stem cell markers at P1 and maintain the expression these markers until P4. Using both techniques, to compare the values of mesenchymal stem cell markers expression at P1 to P4 there was no significant difference. This study indicates that respiratory epithelial cells derived from nasal turbinate retain some of mesenchymal stem cells properties even after serial passages. Both methods of Immunophenotyping are comparable.

El epitelio respiratorio es la primera línea de contacto con los peligros externos. Por lo tanto, puede ser dañado y necesita ser reemplazado para evitar uan cicatrización por fibrosis. La ingeniería de tejidos traqueales es una modalidad de tratamiento alternativo prometedora. Los marcadores de células troncales mesenquimales son proteínas de superficie, que son responsables de algunas propiedades únicas de estas células. El objetivo fue detectar el fenotipo de células troncales mesenquimales entre las células epiteliales respiratorias nasales humanas a través de dos técnicas de inmunofenotipaje. Fueron cultivadas las células epiteliales respiratorias utilizando la técnica de co-cultivo; los fibroblastos se eliminaron en la confluencia dejando solo células epiteliales respiratorias, resultantes de los 4 pasajes. Las células fueron evaluadas para encontrar marcadores de células troncales mesenquimales mediante CD73, CD90, CD105 y el marcador de células troncales hematopoyéticas CD45 en el paso 1 (P1) y el paso 4 (P4), usando citometría de flujo y técnicas de inmunocitoquímica. Las células epiteliales respiratorias expresaron los marcadores de células troncales mesenquimales en P1 y mantuvieron la expresión de estos marcadores hasta P4. No hubo diferencias significativas en el uso de ambas técnicas al comparar los valores de los marcadores de células troncales mesenquimales expresadas desde P1 a P4. Este estudio indica que las células epiteliales respiratorias derivadas de la concha nasal retienen algunas de las propiedades de células troncales mesenquimales, incluso después de pases seriados. Ambos métodos de inmunofenotipificación son comparables.

Cartilage regeneration by chondrogenic induced adult stem cells in osteoarthritic sheep model.

OBJECTIVES: In this study, Adipose stem cells (ADSC) and bone marrow stem cells (BMSC), multipotent adult cells with the potentials for cartilage regenerations were induced to chondrogenic lineage and used for cartilage regenerations in surgically induced osteoarthritis in sheep model. METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of the anterior cruciate ligament and medial meniscus following a 3-weeks exercise regimen. Stem cells from experimental sheep were culture expanded and induced to chondrogenic lineage. Test sheep received a single dose of 2 × 10(7) autologous PKH26-labelled, chondrogenically induced ADSCs or BMSCs as 5 mls injection, while controls received 5 mls culture medium. RESULTS: The proliferation rate of ADSCs 34.4 ± 1.6 hr was significantly higher than that of the BMSCs 48.8 ± 5.3 hr (P = 0.008). Chondrogenic induced BMSCs had significantly higher expressions of chondrogenic specific genes (Collagen II, SOX9 and Aggrecan) compared to chondrogenic ADSCs (P = 0.031, 0.010 and 0.013). Grossly, the treated knee joints showed regenerated de novo cartilages within 6 weeks post-treatment. On the International Cartilage Repair Society grade scores, chondrogenically induced ADSCs and BMSCs groups had significantly lower scores than controls (P = 0.0001 and 0.0001). Fluorescence of the tracking dye (PKH26) in the injected cells showed that they had populated the damaged area of cartilage. Histological staining revealed loosely packed matrixes of de novo cartilages and immunostaining demonstrated the presence of cartilage specific proteins, Collagen II and SOX9. CONCLUSION: Autologous chondrogenically induced ADSCs and BMSCs could be promising cell sources for cartilage regeneration in osteoarthritis.

Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane.

The cornea can be damaged by a variety of clinical disorders or chemical, mechanical, and thermal injuries. The objectives of this study were to induce bone marrow mesenchymal stem cells (BMSCs) to corneal lineage, to form a tissue engineered corneal substitute (TEC) using BMSCs, and to treat corneal surface defects in a limbal stem cell deficiency model. BMSCs were induced to corneal lineage using limbal medium for 10 days. Induced BMSCs demonstrated upregulation of corneal stem cell markers; ß1-integrin, C/EBPδ, ABCG2, and p63, increased protein expression of CK3 and p63 significantly compared with the uninduced ones. For TEC formation, passage 1 BMSCs were trypsinized and seeded on amniotic membrane in a transwell co-culture system and were grown in limbal medium. Limbal stem cell deficiency models were induced by alkaline injury, and the TEC was implanted for 8 weeks. Serial slit lamp evaluation revealed remarkable improvement in corneal regeneration in terms of corneal clarity and reduced vascularization. Histologic and optical coherence tomography analyses demonstrated comparable corneal thickness and achieved stratified epithelium with a compact stromal layer resembling that of normal cornea. CK3 and p63 were expressed in the newly regenerated cornea. In conclusion, BMSCs can be induced into corneal epithelial lineage, and these cells are viable for the formation of TEC, to be used for the reconstruction of the corneal surface in the limbal stem cell deficient model.

Neural-differentiated mesenchymal stem cells incorporated into muscle stuffed vein scaffold forms a stable living nerve conduit.

Autologous nerve grafts to bridge nerve gaps have donor site morbidity and possible neuroma formation resulting in development of various methods of bridging nerve gaps without using autologous nerve grafts. We have fabricated an acellular muscle stuffed vein seeded with differentiated mesenchymal stem cells (MSCs) as a substitute for nerve autografts. Human vein and muscle were both decellularized by liquid nitrogen immersion with subsequent hydrolysis in hydrochloric acid. Human MSCs were subjected to a series of treatments with a reducing agent, retinoic acid, and a combination of trophic factors. The differentiated MSCs were seeded on the surface of acellular muscle tissue and then stuffed into the vein. Our study showed that 35-75% of the cells expressed neural markers such as S100b, glial fibrillary acidic protein (GFAP), p75 NGF receptor, and Nestin after differentiation. Histological and ultra structural analyses of muscle stuffed veins showed attachment of cells onto the surface of the acellular muscle and penetration of the cells into the hydrolyzed fraction of muscle fibers. We implanted these muscle stuffed veins into athymic mice and at 8 weeks post-implantation, the acellular muscle tissue had fully degraded and replaced with new matrix produced by the seeded cells. The vein was still intact and no inflammatory reactions were observed proving the biocompatibility and biodegradability of the conduit. In conclusion, we have successfully formed a stable living nerve conduit which may serve as a substitute for autologous nerves.