Specific Differentiation Media Research Articles

PMSC DIFFERENTIATION INDUCED BY MUSCULOSKELETAL MATRIX: IN VITRO STUDY

Periosteal mesenchymal stem cells (PMSC) are an emerging niche of stem cells to enhance bone healing by tissue engineering process. They have to be differentiated into osteoprogenitors in order to synthesize new bone matrix. In vitro differentiation with specific differentiation medium (DM) is not exactly representative of what occurs in vivo. The interaction between PMSC and growth factors (GF) present in biological matrix is somewhat less understood. The goal of this study is to explore the possibility of spontaneous PMSC differentiation in contact with different biological matrices without DM.500.000 porcine PMSC were seeded on 6-well plates and cultured with proliferation medium (PM). When reaching 80% confluence, biological samples (n=3) of demineralized bone matrix (DBM), decellularized porcine bone allograft (AOp), human bone allograft (AOh), human periosteum (HP) and human fascia lata (HFL) were added. Negative and positive control wells included cells with only PM or DM, respectively. The differentiation progress was assessed by Alizarin Red staining at days 7, 14 and 21. Bone morphogenetic protein content (BMP 2, 4, 5, 6, 7, 8, 9 and 11) of each sample was also investigated by western blot.Alizarin red highlighted bone nodules neoformation on wells containing AOp, AOh and DBM, like positive controls. HP and HFL wells did not show any nodules. These results are correlated to a global higher BMP expression profile in AOp than in HP and HFL but not statistically significant (p=0.38 and p>.99, respectively). The highest expression in each tissue was that of BMP2 and BMP7, which play an important role in osteoinduction.PMSC are well known to participate to bone formation but, despite BMP presence in HP and HFL, they did not permit to achieve osteogenesis alone. The bone contact seems to be essential to induce in vitro differentiation into osteoprogenitors.

Culture Conditions for Human Induced Pluripotent Stem Cell-Derived Schwann Cells: A Two-Centre Study

Adult human Schwann cells represent a relevant tool for studying peripheral neuropathies and developing regenerative therapies to treat nerve damage. Primary adult human Schwann cells are, however, difficult to obtain and challenging to propagate in culture. One potential solution is to generate Schwann cells from human induced pluripotent stem cells (hiPSCs). Previously published protocols, however, in our hands did not deliver sufficient viable cell numbers of hiPSC-derived Schwann cells (hiPSC-SCs). We present here, two modified protocols from two collaborating laboratories that overcome these challenges. With this, we also identified the relevant parameters to be specifically considered in any proposed differentiation protocol. Furthermore, we are, to our knowledge, the first to directly compare hiPSC-SCs to primary adult human Schwann cells using immunocytochemistry and RT-qPCR. We conclude the type of coating to be important during the differentiation process from Schwann cell precursor cells or immature Schwann cells to definitive Schwann cells, as well as the amounts of glucose in the specific differentiation medium to be crucial for increasing its efficiency and the final yield of viable hiPSC-SCs. Our hiPSC-SCs further displayed high similarity to primary adult human Schwann cells.

A Functional in Vitro 3D Model of Human Hematopoietic Niches

Niches in the bone marrow (BM) regulate hematopoietic stem, progenitor and precursor cell fate and behaviour through cell-cell interactions and soluble factor secretion. Niche biology and niche-hematopoietic crosstalk are very complex processes not completely elucidated yet, due essentially to human material accessibility difficulties and limits of existing in vitro culture models. Here, we develop a 3D fully human bone marrow model recapitulating all hematopoietic and non-hematopoietic niche elements to aid further investigation of normal and pathological haematopoiesis and to move away from expensive, time-consuming and imperfect animal models. At first, we have developed a protocol to in vitro production and culture of all human main cellular non-hematopoietic components of bone marrow microenvironment. This comprehensive stroma is generated from human primary cells from femoral head, without any external cell lines contribution, and it reflects accurately the characteristics of the donor. In a 2-step culture process, mesenchymal stromal cells and endothelial cells are first selected and amplified simultaneously in an amplification medium, then they are cultured in the amplification medium as control or in a specific differentiation medium patented by the authors in conventional 2D culture plates or in 3D cultures: in spheroids, by self-organization in ultra-low-adherence plates or on a biomaterial (β-TriCalcium Phosphate), in the U-CUP perfused bioreactor. The resulting cellular stroma composition is analysed by quantifying the gene expression of markers of the osteoblastic (RUNX2, OSX, BSP), adipocytic (LPL, PPARγ, ADIPOQ) and endothelial (CD31) lineages . Moreover, specific markers of hematopoietic niche as ANG1, SCF, CXCL12, LEPR, VCAM, OPN are investigated by RT-qPCR. The spatial organization and the proportion of the different cellular stroma components are analysed by immunofluorescence (Osteoblastic cells: OSX marker, adipocytic cells: Bodipy probe, endothelial cells: CD31). The resulting tissues exhibited all compositional and structural features of human physiological BM: all stroma cells are presents and they express the main niche soluble factors. So, we explore if medullary stroma differentiated using our protocol is able to support the maintenance of HSPCs, to do this we performed coculture experiments in 2D with CD34+ cells sorted from umbilical cord blood. After 14 and 21 days of coculture, hematopoietic cells are recovered, counted and analysed by flow cytometry. Results show that our differentiated stroma is capable of maintaining, expanding and functionally regulated healthy human cord blood-derived HSPCs. An immature compartment (CD34+CD38-) is still present at day 21. Importantly, our primary in vitro niche model supports HSPCs with no cytokine addition. The same results are obtained with healthy human peripheral blood-derived HSPCs cocultured in 3D with the stroma organised in spheroids. Moreover, we demonstrated that our BM model is capable not only to maintain HSPC compartment but also to drive hematopoietic differentiation. In 2D coculture we detected mature blood cells and in 3D cultures, peripheral blood-derived HSPCs cocultured with spheroids are able to engage in erythroid lineage and recapitulate the early phases of erythropoiesis. These results demonstrate the fully complexity and huge versatility of our functional in vitro 3D model of human hematopoietic niches. Our exhaustive fully human hematopoietic niche model prepared from the primary cells of single donor, enables the design of advanced, tuneable in vitro BM proxies for the study of human physiological and pathological haematopoiesis and for screening molecules for predictive purposes in personalized medicine.

Isolation and Characterization of Stem Cells from the Anal Canal Transition Zone in Pigs

BackgroundUtilization of autologous stem cells has been proposed for the treatment of anal incontinence despite a lack of understanding of their mechanism of action and of the physiological healing process of anal sphincters after injury.AimsWe aim to develop a technique allowing isolation and further study of local mesenchymal stem cells, directly from anal canal transition zone in pig.MethodsAnal canal was resected “en bloc” from two young pigs and further microdissected. The anal canal transition zone was washed and digested with 0.1% type I collagenase for 45 min at 37 °C. The isolated cells were plated on dishes in mesenchymal stem cell medium and trypsinized when confluent. Cells were further used for flow cytometry analysis and differentiation assays.ResultsThe anal canal transition zone localization was confirmed with H&E staining. Following culture, cells exhibited a typical “fibroblast-like” morphology typical of stem cells. Isolated cells were positive for CD90 and CD44 but negative for CD14, CD34, CD45, CD105, CD106, and SLA-DR. Following incubation with specific differentiation medium, isolated cells differentiated into adipocytes, osteoblasts, and chondrocytes, confirming in vitro multipotency.ConclusionsHerein, we report for the first time the presence of mesenchymal stem cells in the anal canal transition zone in pigs and the feasibility of their isolation. This preliminary study opens the path to the isolation of human anal canal transition zone mesenchymal stem cells that might be used to study sphincters healing and to treat anal incontinence.

Abstract 111: Head and neck cancer cells can differentiate and resemble their tissue of origin

Abstract Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease with a mortality rate of about 50%. It represents the sixth most common type of malignant tumor in the world. Risk factors include tobacco and alcohol usage and infection with the human papilloma virus (HPV). HPV-negative HNSCCs frequently display areas of cornification evident by keratin pearls in the tumor tissue. Cornification represents a natural differentiation path of keratinocytes in the normal epidermis and oral mucosa. To investigate the mechanisms of HNSCC cell differentiation we have established spheroid cell cultures from patient-derived HNSCC and parotid gland adenoid cystic carcinoma (ACC) tissue that grow in suspension under serum-free conditions. The use of a specific differentiation medium induced striking adhesion, loss of proliferation, and differentiation in tumor cells. Spheroid cells grew as single cell clones under serum-free conditions with a cloning efficiency of 40-60%, which was fully diminished under differentiating conditions. HNSCC cells cornified as indicated by the formation of lamellar bodies in the cytoplasm of adherent cells and an upregulation of cornification markers SPRR3 and involucrin. ACC cells upregulated parotid gland differentiation markers including α-amylase. RNA-seq analysis in HNSCC cells confirmed an upregulation of signaling pathways associated with cornification and epithelial cell differentiation. Conversely, pathways regulating the three-dimensional organization of the genome were downregulated upon differentiation. This was accompanied by the formation of ATRX-positive heterochromatin foci in the nucleus of differentiated ACC and HNSCC cells resembling those previously described to arise during therapy-induced senescence. Moreover, gas chromatography mass spectrometry analysis revealed a lack of essential amino acids including leucine to be implicated in the differentiation process. Altogether, our spheroid model of HNSCC and ACC cells is suitable to analyze the mechanisms underlying tumor cell differentiation and might lead to new therapeutic approaches that can drive long-term repopulating HNSCC and ACC cells into differentiation. Citation Format: Felix Oppel, Senyao Shao, Sarah Gendreizig, Philipp Kühnel, Vivien Przybycin, Carsten Hain, Pascal Schmidt, Matthias Schürmann, Peter Goon, Karsten Niehaus, Jörn Kalinowski, Holger Sudhoff. Head and neck cancer cells can differentiate and resemble their tissue of origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 111.

Metformin and Vitamin D Modulate Inflammation and Autophagy during Adipose-Derived Stem Cell Differentiation.

Adipose-derived stem cells (ADSCs) came out from the regenerative medicine landscape for their ability to differentiate into several phenotypes, contributing to tissue regeneration both in vitro and in vivo. Dysregulation in stem cell recruitment and differentiation during adipogenesis is linked to a chronic low-grade inflammation and macrophage infiltration inside the adipose tissue, insulin resistance, cardiovascular disease and obesity. In the present paper we aimed to evaluate the role of metformin and vitamin D, alone or in combination, in modulating inflammation and autophagy in ADSCs during adipogenic commitment. ADSCs were cultured for 21 days in the presence of a specific adipogenic differentiation medium, together with metformin, or vitamin D, or both. We then analyzed the expression of FoxO1 and Heat Shock Proteins (HSP) and the secretion of proinflammatory cytokines IL-6 and TNF-α by ELISA. Autophagy was also assessed by specific Western blot analysis of ATG12, LC3B I, and LC3B II expression. Our results showed the ability of the conditioned media to modulate adipogenic differentiation, finely tuning the inflammatory response and autophagy. We observed a modulation in HSP mRNA levels, and a significant downregulation in cytokine secretion. Taken together, our findings suggest the possible application of these molecules in clinical practice to counteract uncontrolled lipogenesis and prevent obesity and obesity-related metabolic disorders.

BEHAVIOR OF POPULATIONS OF MESENCHYMAL STEM CELLS OBTAINED FROM THE PULP OF MOUSE INCISORS

Background Mesenchymal stem cells (MSCs) are potential candidates for tissue engineering and regenerative medicine applications. Stromal tissues, from which MSCs are extracted, exist all over the body, suggesting that their precursors are locally available in multiple organs, providing a source of MSCs for homeostasis and repair of tissues. Teeth are one of the organs that contain MSCs. The understanding of properties of the different resident dental-derived stem cell populations may lead to the development of biologically based dental therapies. The continuously growing mouse incisor provides a highly accessible model to study stem cell behavior. Therefore, considering the importance of clinical applications of MSCs obtained from dental pulp, an experimental animal model was developed to study the behavior of different populations of MSCs. Methods Incisors from 7-day postnatal mice were extracted from the mandible and maxilla and pulp tissue was dissociated using collagenase / dispase. The cell suspension was incubated to 80% confluence. The RNA was extracted from the cells, quantified, and transcribed into cDNA. For gene expression analysis (qPCR), MSCs clusters of differentiation genes (CD90, CD73, CD105) were used. For differentiation analysis, cells were incubated for 21 days in specific differentiation media (osteogenic, adipogenic, and chondrogenic) and analysed by immunocytochemistry. Flow cytometry (FACS) was also performed to sort CD90 positive cells. Thus, the newly dissociated cell suspension was incubated with anti-CD90 antibody. The FACS sorted cells were cultured until confluence and were again analysed by qPCR. Results MSCs obtained from the pulp of mouse incisors presented the minimum criteria for the definition of multipotent MSCs, that is, they expressed CD90, CD73, and CD105 and differentiated in vitro into three cell lineages. However, clusters of differentiation genes were more remarkable for CD90 positive cells than for non-FACS sorted cells. This is an indication that, among the MSC populations of the incisor pulp, there are different potentials for cell differentiation. Conclusions The continuously growing mouse incisor has in its pulp tissue different niches and populations of stem cells that have different behavior regarding the potential for differentiation.

Osteogenic and chondrogenic differentiation potential of mesenchymal stem cells obtained from the bone marrow and placenta

Mesenchymal stem cells (MSC) represent a perspective resource for cell biotechnology. However the question of chondrogenic and osteogenic capacity of MSC of different origin remains under study.The aim of this study was to analyze the osteo-chondrogenic differentiation potential of MSC obtained from the bone marrow and placenta. The results of our studies have indicated that bone marrow-derived and placenta-derived MSC showed a chondrogenic potential in vitro after a chondrogenic induction with specific differentiation media. But for bone marrowderived MSC, the chondrogenic program was realized by expression of collagens (Coll2, Coll10), while in placenta-derived MSC cultures we found a progressive increase in COMP and Ver expression, so bone marrow-derived MSC is more preferable for use in cartilage tissue engineering. Regarding the results on alkaline phosphatase and alizarin red staining, bone marrowderived MSC showed a more significant osteogenic potential compared to placenta-derived MSC. Bone marrow-derived MSC in the composition of fibrin gel after osteogenic induction on the 14th day exhibited the activity of alkaline phosphatase, calcium depositions inside the cells and extracellular matrix, the increase in Sp7 and DMP expression.

24: Deceased Donor Adipose Tissue: An Untapped Source of Mesenchymal Stem Cells

Purpose: Adipose tissue from living donors is a widely accepted source of mesenchymal stem cells. Adipose tissue can also be procured from deceased donors in high volumes and routinely during the deceased donor workup. Materials: Adipose tissue was excised from the abdomen of research-consented deceased donors. Sections of the adipose tissue were mechanically minced and washed with saline. The mixture was then filtered, centrifuged, and resuspended in wash buffer to represent the stromal vascular fraction (SVF). The SVF was tested for viability, total nucleated cell count (TNC), and the presence of mesenchymal stem cells (MSC). The functionality of the MSC was assessed by their growth potential and their capacity to differentiate using specific differentiation media. Results: We observed that the mean TNC/g of samples processed fell above the living donor value ranges. The mean %MSC/TNC value fell within the living donor value range (Table 1). Induction of adipocyte differentiation led to the morphological conversion of MSC to form lipid droplets, a characteristic of mature white adipocytes, accompanied by the expression of adipocyte differentiation genes. Similarly, differentiation of MSC into osteocytes and chondrocytes, when exposed to the respective differentiation media, was observed. Differentiation specific gene expression was also confirmed. In all cases, the undifferentiated cells did not exhibit any changes either at the morphological or gene expression level (Figure 1). Conclusion: The deceased donor stem cells can be routinely procured and potentially supplement the current available living donor stem cells sources. We have established that deceased donor adipose-derived MSC are similar to living donor adipose-derived MSC and can differentiate to adipocytes, osteocytes, and chondrocytes. Figure 1. Differentiation of MSC isolated from non-enzyme treated SVF. Light microscopic images are representative of three separate experiments. (A) Adipocyte differentiation. (C) Osteocyte differentiation. (E) Chondrocyte differentiation. (B,D,F) Semi-quantitative qRT-PCR analysis of lineage-specific gene expression.

Isolation and Differentiation of Mesenchymal Stem Cells From Broiler Chicken Compact Bones

Chicken mesenchymal stem cells (MSCs) can be used as an avian culture model to better understand osteogenic, adipogenic, and myogenic pathways and to identify unique bioactive nutrients and molecules which can promote or inhibit these pathways. MSCs could also be used as a model to study various developmental, physiological, and therapeutic processes in avian and other species. MSCs are multipotent stem cells that are capable of differentiation into bone, muscle, fat, and closely related lineages and express unique and specific cell surface markers. MSCs have been isolated from numerous sources including human, mouse, rabbit, and chicken with potential clinical and agricultural applications. MSCs from chicken compact bones have not been isolated and characterized yet. In this study, MSCs were isolated from compact bones of the femur and tibia of day-old male broiler chicks to investigate the biological characteristics of the isolated cells. Isolated cells took 8–10 days to expand, demonstrated a monolayer growth pattern and were plastic adherent. Putative MSCs were spindle-shaped with elongated ends and showed rapid proliferation. MSCs demonstrated osteoblastic, adipocytic, and myogenic differentiation when induced with specific differentiation media. Cell surface markers for MSCs such as CD90, CD105, CD73, CD44 were detected positive and CD31, CD34, and CD45 cells were detected negative by PCR assay. The results suggest that MSCs isolated from broiler compact bones (cBMSCs) possess similar biological characteristics as MSCs isolated from other chicken tissue sources.

The Osteogenic and Tenogenic Differentiation Potential of C3H10T1/2 (Mesenchymal Stem Cell Model) Cultured on PCL/PLA Electrospun Scaffolds in the Absence of Specific Differentiation Medium

The differentiation potential of mesenchymal stem cells (MSC) has been extensively tested on electrospun scaffolds. However, this potential is often assessed with lineage-specific medium, making it difficult to interpret the real contribution of the properties of the scaffold in the cell response. In this study, we analyzed the ability of different polycaprolactone/polylactic acid PCL/PLA electrospun scaffolds (pure or blended compositions, random or aligned fibers, various fiber diameters) to drive MSC towards bone or tendon lineages in the absence of specific differentiation medium. C3H10T1/2 cells (a mesenchymal stem cell model) were cultured on scaffolds for 96 h without differentiation factors. We performed a cross-analysis of the cell–scaffold interactions (spreading, organization, and specific gene expression) with mechanical (elasticity), morphological (porosity, fibers diameter and orientation) and surface (wettability) characterizations of the electrospun fibers. We concluded that (1) osteogenic differentiation can be initiated on pure PCL-based electrospun scaffolds without specific culture conditions; (2) fiber alignment modified cell organization in the short term and (3) PLA added to PCL with an increased fiber diameter encouraged the stem cells towards the tendon lineage without additional tenogenic factors. In summary, the differentiation potential of stem cells on adapted electrospun fibers could be achieved in factor-free medium, making possible future applications in clinically relevant situations.

Human dental pulp pluripotent-like stem cells promote wound healing and muscle regeneration

BackgroundDental pulp represents an easily accessible autologous source of adult stem cells. A subset of these cells, named dental pulp pluripotent-like stem cells (DPPSC), shows high plasticity and can undergo multiple population doublings, making DPPSC an appealing tool for tissue repair or maintenance.MethodsDPPSC were harvested from the dental pulp of third molars extracted from young patients. Growth factors released by DPPSC were analysed using antibody arrays. Cells were cultured in specific differentiation media and their endothelial, smooth and skeletal muscle differentiation potential was evaluated. The therapeutic potential of DPPSC was tested in a wound healing mouse model and in two genetic mouse models of muscular dystrophy (Scid/mdx and Sgcb-null Rag2-null γc-null).ResultsDPPSC secreted several growth factors involved in angiogenesis and extracellular matrix deposition and improved vascularisation in all three murine models. Moreover, DPPSC stimulated re-epithelialisation and ameliorated collagen deposition and organisation in healing wounds. In dystrophic mice, DPPSC engrafted in the skeletal muscle of both dystrophic murine models and showed integration in muscular fibres and vessels. In addition, DPPSC treatment resulted in reduced fibrosis and collagen content, larger cross-sectional area of type II fast-glycolytic fibres and infiltration of higher numbers of proangiogenic CD206+ macrophages.ConclusionsOverall, DPPSC represent a potential source of stem cells to enhance the wound healing process and slow down dystrophic muscle degeneration.

Isolation, culture and characterization of multipotent mesenchymal stem cells from goat umbilical cord blood

ABSTRACT: Mesenchymal stem cells (MSC) reside in small numbers in many adult tissues and organs, and play an active role in the homeostasis of these sites. Goat derived multipotent MSC have been established from bone marrow, adipose tissues and amniotic fluid. Umbilical cord blood (UCB) is considered an important source of these cells. However, the MSC isolation from the goat UCB has not been demonstrated. Therefore, the aim of the present study was to isolate, culture and characterize goat umbilical cord blood derived mesenchymal stem cells. MSC were isolated from UCB by Ficoll-Paque density centrifugation and cultured in DMEM supplemented with 10% or 20% FBS. FACS analysis was performed and induction lineage differentiation was made to characterize these cells. They exhibited two different populations in flow cytometry, and revealed the positive expression of CD90, CD44 and CD105, but negative staining for CD34 in larger cells, and positive stained for CD90 and CD105, but negative for CD44 and CD34 in the smaller cells. MSC from goat UCB showed capability to differentiate into chondrocytes and osteoblasts when incubated with specific differentiation medium. Present study established that goat mesenchymal stem cells can be derived successfully from umbilical cord blood.

Biodegradable polyester networks including hydrophilic groups favor BMSCs differentiation and can be eroded by macrophage action

The aim of this study is to show that introducing a small fraction of hydrophilic groups into a hydrophobic polyester favor the macrophage activity by accelerating the degradation action in aqueous media. It is also seen that differentiation of MSCs cultured in monolayer towards bone in specific differentiation media is favored in these materials with respect to the corresponding pristine polyesters. Polymer networks based in polycarpolactone or poly(l-lactide) and containing a small fraction of poly(hydroxyethyl acrylate) have been synthesized. Degradation kinetics in vitro was monitored by mass loss and swelling capacity of the polymer network in good solvents, the later as representative of chain cleavage. Hydrolytic and enzymatic degradation is accelerated by the inclusion of poly(hydroxyethyl acrylate) blocks in the network. Macrophages were cultured on the surface of the network films, showing its capacity to erode the material surface but also to accelerate bulk degradation. Bone marrow mesenchymal stem cells were cultured in monolayer on the membranes in osteogenic media, showing an increase of specific markers expression in comparison to pristine polyesters.

Silk-ionomer and silk-tropoelastin hydrogels as charged three-dimensional culture platforms for the regulation of hMSC response.

The response of human bone marrow-derived mesenchymal stem cells (hMSCs) encapsulated in three-dimensional (3D) charged protein hydrogels was studied. Combining silk fibroin (S) with recombinant human tropoelastin (E) or silk ionomers (I) provided protein composite alloys with tunable physicochemical and biological features for regulating the bioactivity of encapsulated hMSCs. The effects of the biomaterial charges on hMSC viability, proliferation and chondrogenic or osteogenic differentiation were assessed. The silk-tropoelastin or silk-ionomers hydrogels supported hMSC viability, proliferation and differentiation. Gene expression of markers for chondrogenesis and osteogenesis, as well as biochemical and histological analysis, showed that hydrogels with different S/E and S/I ratios had different effects on cell fate. The negatively charged hydrogels upregulated hMSC chondrogenesis or osteogenesis, with or without specific differentiation media, and hydrogels with higher tropoelastin content inhibited the differentiation potential even in the presence of the differentiation media. The results provide insight on charge-tunable features of protein-based biomaterials to control hMSC differentiation in 3D hydrogels, as well as providing a new set of hydrogels for the compatible encapsulation and utility for cell functions. Copyright © 2016 John Wiley & Sons, Ltd.

Hepatogenic differentiation from human adipose-derived stem cells and application for mouse acute liver injury

Adipose-derived stem cells (ADSCs) derived from adipose tissue have the capacity to differentiate into endodermal, mesoderm, and ectodermal cell lineages in vitro, which are an ideal engraft in tissue-engineered repair. In this study, human ADSCs were isolated from subcutaneous fat. The markers of ADSCs, CD13, CD71, CD73, CD90, CD105, CD166, CYP3A4, and ALB were detected by immunofluorescence assays. Human ADSCs were cultured in a specific hepatogenesis differentiation medium containing HGF, bFGF, nicotinamide, ITS, and oncostatin M for hepatogenic differentiation. The hepatocyte markers were analyzed using immunofluorescence and real-time PCR after dramatic changes in morphology. Hepatocytes derived from ADSCs or ADSCs were transplanted into the mice of liver injury for observation cells colonization and therapy in liver tissue. The result demonstrated that human ADSCs were positive for the CD13, CD71, CD73, CD90, CD105, and CD166 but negative for hepatocyte markers, ALB and CYP3A4. After hepatogenic differentiation, the hepatocytes were positive for liver special markers, gene expression level showed a time-lapse increase with induction time. Human ADSCs or ADSCs-derived hepatocyte injected into the vein could improve liver function repair and functionally rescue the CCl4-treated mice with liver injury, but the ADSCs transplantation was better than ADSCs-derived hepatocyte transplantation. In conclusion, our research shows that a population of hepatocyte can be specifically generated from human ADSCs and that cells may allow for participation in tissue-repair.

In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells.

Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone mar- row have shown a homogenous population of rare stage-specific embryonic antigen 1 (SSEA-1) positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells (ISCs) in order to investigate their differentiation potential for future use in cell therapy. This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting (MACS) followed by characteriza- tion with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone (DTZ) staining was use, followed by reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA. The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 (OCT-4) detected by immunocytochem- istry and C-X-C chemokine receptor type 4 (CXCR4) and stem cell antigen-1 (SCA-1) detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 (pancreatic transcription factors), Ngn3 (endocrine progenitor marker), Insulin1 and Insulin2 (pancreaticβ-cell markers). Additionally, our results demonstrate expression of Pdx1 and Glut2 protein and insulin secretion in response to a glucose challenge in the differentiated cells. Our study clearly demonstrates the potential of SSEA-1 positive cells to differentiate into insulin secreting cells in defined culture conditions for clinical ap- plications.

Cardiac Adipose-Derived Stem Cells Exhibit High Differentiation Potential to Cardiovascular Cells in C57BL/6 Mice.

Adipose-derived stem cells (AdSCs) have recently been shown to differentiate into cardiovascular lineage cells. However, little is known about the fat tissue origin-dependent differences in AdSC function and differentiation potential. AdSC-rich cells were isolated from subcutaneous, visceral, cardiac (CA), and subscapular adipose tissue from mice and their characteristics analyzed. After four different AdSC types were cultured with specific differentiation medium, immunocytochemical analysis was performed for the assessment of differentiation into cardiovascular cells. We then examined the in vitro differentiation capacity and therapeutic potential of AdSCs in ischemic myocardium using a mouse myocardial infarction model. The cell density and proliferation activity of CA-derived AdSCs were significantly increased compared with the other adipose tissue-derived AdSCs. Immunocytochemistry showed that CA-derived AdSCs had the highest appearance rates of markers for endothelial cells, vascular smooth muscle cells, and cardiomyocytes among the AdSCs. Systemic transfusion of CA-derived AdSCs exhibited the highest cardiac functional recovery after myocardial infarction and the high frequency of the recruitment to ischemic myocardium. Moreover, long-term follow-up of the recruited CA-derived AdSCs frequently expressed cardiovascular cell markers compared with the other adipose tissue-derived AdSCs. Cardiac adipose tissue could be an ideal source for isolation of therapeutically effective AdSCs for cardiac regeneration in ischemic heart diseases. Significance: The present study found that cardiac adipose-derived stem cells have a high potential to differentiate into cardiovascular lineage cells (i.e., cardiomyocytes, endothelial cells, and vascular smooth muscle cells) compared with stem cells derived from other adipose tissue such as subcutaneous, visceral, and subscapular adipose tissue. Notably, only a small number of supracardiac adipose-derived stem cells that were systemically transplanted sufficiently improved cardiac functional recovery after myocardial infarction, differentiating into cardiovascular cells in the ischemic myocardium. These findings suggest a new autologous stem cell therapy for patients with myocardial ischemia, especially those with secondary myocardial ischemia after cardiovascular open chest surgery.

Expression analysis of Notch signaling pathway molecules in SHED cultured in keratinocyte growth medium

Aim: To detect the expression of molecules associated with Notch signaling pathway in stem cells from human exfoliated deciduous teeth (SHED) cultured in specific differentiation medium, namely, keratinocyte growth medium (KGM). Methods: RNA was extracted from SHED harvested on day 1, 3 and 7. RNA was reverse-transcribed to obtain the cDNA and then proceeded with PCR using specific primers for the Notch signaling pathway molecules (Notch1, Jagged-1, Jagged-2 and, Hes1) as well as stem cell marker (Nanog). PCR products were electrophoresed on a 2% agarose gel and stained with SYBR green. Results: Notch-1 was highly expressed in SHED cultured in KGM and showed increase in density as the days progressed, while Jagged-1 showed a decrease. Jagged-2 on the other hand, showed a slight increase on day 3 followed by a decrease on day 7. However, Hes-1 was not expressed in SHED cultured in KGM. Nanog showed expression only on day 3 and gradually increased in expression on day 7. Conclusions: Notch signaling pathway associated molecules; Notch-1, Jagged-1, Jagged-2, and stem cell marker Nanog are expressed in SHED cultured in KGM which may be involved in the differentiation into epithelial-like cells in human dental pulp tissues.

Formation of cartilage and synovial tissue by human gingival stem cells.

Human gingival stem cells (HGSCs) can be easily isolated and manipulated in culture to investigate their multipotency. Osteogenic differentiation of bone-marrow-derived mesenchymal stem/stromal cells has been well documented. HGSCs derive from neural crests, however, and their differentiation capacity has not been fully established. The aim of the present report was to investigate whether HGSCs can be induced to differentiate to osteoblasts and chondrocytes. HGSCs were cultured either in a classical monolayer culture or in three-dimensional floating micromass pellet cultures in specific differentiation media. HGSC differentiation to osteogenic and chondrogenic lineages was determined by protein and gene expression analyses, and also by specific staining of cells and tissue pellets. HGSCs cultured in osteogenic differentiation medium showed induction of Runx2, alkaline phosphatase (ALPL), and osterix expression, and subsequently formed mineralized nodules consistent with osteogenic differentiation. Interestingly, HGSC micromass cultures maintained in chondrogenic differentiation medium showed SOX9-dependent differentiation to both chondrocyte and synoviocyte lineages. Chondrocytes at different stages of differentiation were identified by gene expression profiles and by histochemical and immunohistochemical staining. In 3-week-old cultures, peripheral cells in the micromass cultures organized in layers of cuboidal cells with villous structures facing the medium. These cells were strongly positive for cadherin-11, a marker of synoviocytes. In summary, the findings indicate that HGSCs have the capacity to differentiate to osteogenic, chondrogenic, and synoviocyte lineages. Therefore, HGSCs could serve as an alternative source for stem cell therapies in regenerative medicine for patients with cartilage and joint destructions, such as observed in rheumatoid arthritis.