Tailored generation of insulin producing cells from canine mesenchymal stem cells derived from bone marrow and adipose tissue

The trend of regenerative therapy for diabetes in human and veterinary practices has conceptually been proven according to the Edmonton protocol and animal models. Establishing an alternative insulin-producing cell (IPC) resource for further clinical application is a challenging task. This study investigated IPC generation from two practical canine mesenchymal stem cells (cMSCs), canine bone marrow-derived MSCs (cBM-MSCs) and canine adipose-derived MSCs (cAD-MSCs). The results illustrated that cBM-MSCs and cAD-MSCs contain distinct pancreatic differentiation potential and require the tailor-made induction protocols. The effective generation of cBM-MSC-derived IPCs needs the integration of genetic and microenvironment manipulation using a hanging-drop culture of PDX1-transfected cBM-MSCs under a three-step pancreatic induction protocol. However, this protocol is resource- and time-consuming. Another study on cAD-MSC-derived IPC generation found that IPC colonies could be obtained by a low attachment culture under the three-step induction protocol. Further, Notch signaling inhibition during pancreatic endoderm/progenitor induction yielded IPC colonies through the trend of glucose-responsive C-peptide secretion. Thus, this study showed that IPCs could be obtained from cBM-MSCs and cAD-MSCs through different induction techniques. Also, further signaling manipulation studies should be conducted to maximize the protocol’s efficiency.

Tailor-made generation of insulin-producing cells from canine mesenchymal stem cells derived from bone marrow and adipose tissue

Trend of regenerative therapy for diabetes in human and veterinary practice has conceptually been proven according to Edmonton protocol and animal models. Establishing an alternative insulin-producing cell (IPC) resource is a challenge task for further clinical application. In this study, IPC generation from two practical canine mesenchymal stem cells(cMSCs), canine bone marrow-derived MSCs (cBM-MSCs) and canine adipose-derived MSCs(cAD-MSCs), was of interest. The results illustrated that cBM-MSCs and cAD-MSCs contained distinct pancreatic differentiation potential and required the tailor-made induction protocols. Effective generation of cBM-MSC-derived IPCs needed an integration of genetic and microenvironment manipulation using hanging-drop culture of PDX-1-transfected cBM-MSCs under three-step pancreatic induction protocol. However, this protocol was resource- and time-consumed. Another study on cAD-MSC-derived IPC generation found that IPC colonies could be obtained by low attachment culture under three-step induction protocol. Further Notch signaling inhibition during pancreatic endoderm/progenitor induction yielded IPC colonies with trend of glucose-responsive C-peptide secretion. Thus, this study showed that IPCs could be obtained from cBM-MSCs and cAD-MSCs by different induction techniques, and further signaling manipulation study should be conducted to maximize the protocol efficiency.

In vitro 3D Spheroid Culture Developed on the Parafilm Surface Using HEK-293 Cells

Preclinical research to predict the effects of drugs and chemicals on humans is commonly carried out either by cell culture studies in vitro condition or on animals in vivo condition. While drug studies tested on cells cultured as a monolayer in plastic flasks are incompatible with realistic results, falsifying findings can also be achieved from in vivo studies performed on different species. In recent years, research on drug tests using spheroid cultures formed by growing cells in three-dimensional (3D) in vitro has attracted great interest. 3D spheroid structures are formed by growing the cells in a drop suspended on superhydrophobic surfaces. In this study, HEK-293 cells were investigated on parafilm surfaces displaying superhydrophobic properties by growing in 2 µl volume using hanging drop culture method in terms of spheroid formation. Light microscopy images from spheroid structures were taken on different incubation days and the area of spheroids was measured using the ImageJ program. Our study holds important findings for a chip platform that can be developed for use in vitro drug tests.

A Dynamic Hanging-Drop System for Mesenchymal Stem Cell Culture

There have been many microfluid technologies combined with hanging-drop for cell culture gotten developed in the past decade. A common problem within these devices is that the cell suspension introduced at the central inlet could cause a number of cells in each microwell to not regularize. Also, the instability of droplets during the spheroid formation remains an unsolved ordeal. In this study, we designed a microfluidic-based hanging-drop culture system with the design of taper-tube that can increase the stability of droplets while enhancing the rate of liquid exchange. A ring is surrounding the taper-tube. The ring can hold the cells to enable us to seed an adequate amount of cells before perfusion. Moreover, during the period of cell culture, the mechanical force around the cell is relatively low to prevent stem cells from differentiate and maintain the phenotype. As a result of our hanging system design, cells are designed to accumulate at the bottom of the droplet. This method enhances convenience for observation activities and analysis of experiments. Thus, this microfluid chip can be used as an in vitro platform representing in vivo physiological conditions, and can be useful in regenerative therapy.

Ultrastructural characteristics and immune profile of equine MSCs from fetal adnexa

Both in human and equine species, mesenchymal stem cells (MSCs) from amniotic membrane (AM) and Wharton’s jelly (WJ), may be particularly useful for immediate use or in later stages of life, after cryopreservation in cell bank. The aim of this study was to compare equine AM- and WJ-MSCs in vitro features that may be relevant for their clinical employment. MSCs were more easily isolated from WJ, even if MSCs derived from AM exhibited more rapid proliferation (P < 0.05). Osteogenic and chondrogenic differentiation were more prominent in MSCs derived from WJ. This is also suggested by the lower adhesion of AM cells, demonstrated by the greater volume of spheroids after hanging drop culture (P < 0.05). Data obtained by PCR confirmed the immunosuppressive function of AM and WJ-MSCs and the presence of active genes specific for anti-inflammatory and angiogenic factors (IL-6, IL 8, IL-β1). For the first time, by means of transmission electron microscopy (TEM), we ascertained that equine WJ-MSCs constitutively contain a very impressive number of large vesicular structures, scattered throughout the cytoplasm. Moreover, an abundant extracellular fibrillar matrix was located in the intercellular spaces among WJ-MSCs. Data recorded in this study reveal that MSCs from different fetal tissues have different characteristics that may drive their therapeutic use. These finding could be noteworthy for horses as well as for other mammalian species, including humans.