Construction of functional pancreatic artificial islet tissue composed of fibroblast-modified polylactic-co-glycolic acid membrane and pancreatic stem cells

2017 ◽  
Vol 32 (3) ◽  
pp. 362-372 ◽  
Author(s):  
Liping Liu ◽  
Jing Tan ◽  
Baoyuan Li ◽  
Qian xie ◽  
Junwen Sun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Blood Glucose ◽  
Nude Mice ◽  
Glycolic Acid ◽  
Blood Levels ◽  
Pancreatic Stem Cells ◽  
Short Period ◽  
Islet Tissue

Objective To improve the biocompatibility between polylactic- co-glycolic acid membrane and pancreatic stem cells, rat fibroblasts were used to modify the polylactic- co-glycolic acid membrane. Meanwhile, we constructed artificial islet tissue by compound culturing the pancreatic stem cells and the fibroblast-modified polylactic- co-glycolic acid membrane and explored the function of artificial islets in diabetic nude mice. Methods Pancreatic stem cells were cultured on the fibroblast-modified polylactic- co-glycolic acid membrane in dulbecco's modified eagle medium containing activin-A, β-catenin, and exendin-4. The differentiated pancreatic stem cells combined with modified polylactic- co-glycolic acid membrane were implanted subcutaneously in diabetic nude mice. The function of artificial islet tissue was explored by detecting blood levels of glucose and insulin in diabetic nude mice. Moreover, the proliferation and differentiation of pancreatic stem cells on modified polylactic- co-glycolic acid membrane as well as the changes on the tissue structure of artificial islets were investigated by immunofluorescence and haematoxylin and eosin staining. Results The pancreatic stem cells differentiated into islet-like cells and secreted insulin when cultured on fibroblast-modified polylactic- co-glycolic acid membrane. Furthermore, when the artificial islet tissues were implanted into diabetic nude mice, the pancreatic stem cells combined with polylactic- co-glycolic acid membrane modified by fibroblasts proliferated, differentiated, and secreted insulin to reduce blood glucose levels in diabetic nude mice. Conclusion Pancreatic stem cells can be induced to differentiate into islet-like cells in vitro. In vivo, the artificial islet tissue can effectively regulate the blood glucose level in nude mice within a short period. However, as time increased, the structure of the artificial islets was destroyed due to the erosion of blood cells that resulted in the gradual loss of artificial islet function.

Reevaluation of Factors Leading to the Successful Isolation of Mesenchymal Stem Cells From Human Umbilical Cord Blood and Their Differentiation to Mesenchymal Lineages.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3232-3232
Author(s):  
Xiaohong Zhang ◽  
Masako Hirai ◽  
Susana Cantero ◽  
Rodica Ciubotariu ◽  
Allen Hirsh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Success Rate ◽  
Nude Mice ◽  
Culture Methods ◽  
Successful Isolation ◽  
High Proliferation Rate

Abstract Abstract 3232 Poster Board III-169 Introduction Cord blood derived mesenchymal stem cells (CB-MSC) have been identified as an alternative cell source to bone marrow derived mesenchymal stem cells (BM-MSC) and adipose tissue derived mesenchymal stem cells (AT-MSC) for use in regenerative medicine. However, the low frequency of these cells in cord blood (CB) has led to conflicting reports of its efficacy and this, in turn, has been the main reason limiting their clinical use thus far. We searched for critical factors determining successful isolation of CB-MSC from more than 300 units of CB donated to two public CB banks using a range of different collection methods for CB. We applied several processing and culture methods to identify an optimal method for isolating CB-MSC. Proliferative, in vitro differentiation ability and immunosuppressive ability of CB-MSC were compared with BM- and AT-MSC. CB-MSC cultured with scaffolds were transplanted to nude mice. Additionally, chromosomal stability of CB-MSC after long-term culture was analyzed. Materials and Methods CB was collected after obtaining informed consent at two collection facilities: either while the placenta was in utero, or after the delivery of the placenta (ex utero). The mononuclear cells (MNC) were isolated by Ficoll-Paque (FP) density gradient centrifugation or other methods and subjected to a colony forming unit-fibroblast (CFU-F) assay. Their ability to differentiate into osteoblasts, chondorocytes, and adipocytes was tested in vitro and in vivo. Specific genes for differentiation to the mesoderm lineage were identified by RT-PCR. Immunosuppression by CB-MSC was tested by addition of cells to phytohemagglutinin (PHA) activated human T cells and to mixed lymphocyte reactions. Karyotypes of expanded CB-MSC were analyzed. Osteogenesis and chondrogenesis of CB-MSC in vivo were examined by transplantation of CB-MSC with scaffolds (β-TCP block, collagen sponge) subcutaneously to nude mice. Results CB-MSCs capable of proliferating were isolated from 121 units of 307 units of CB (63.1 ± 20.7 ml w/o anticoagulant). Two critical factors contributing to the success rate of isolating CB-MSC were: interval between collection of CB and processing of cells, and CB volume. When the interval was less than 2 hours there was a marked increase in success, S, according to the equation S=0.55*t-0.4316, (R2>0.99, n=81). There was also a more modest increase in S from increasing volume: S=0.0034*V (ml) + 0.2244, (R2>0.85, n=249). When both volume was higher than 90 ml and time was shorter than 5 hours, the success rate increased to 84.6%. The mean number of clonies from the units was calculated to be 1.59 ± 1.48 CFU /108 MNC (n=40) and 2.7 ± 2.3 CFU/CB unit. Variation in isolation and culture methods of did not improve the success rate. Most CB-MSC isolated grew rapidly and proliferated at more than 40 PDL (>15 passages), whereas BM-MSC and AT-MSC stopped proliferating at about 10 PDL. The CB-MSC showed higher differentiation ability to chondrocytes more than BM-MSC and AT-MSC. In vivo osteogenesis and chondrogenesis were observed when CB-MSC cultured with scaffolds were transplanted subcutaneously to nude mice. CB-MSC suppressed proliferation of lymphocytes stimilated allogeneically (mixed lymphocyte reaction) and by PHA as the dose of cells increased similar to finding with BM-MSC and AT-MSC. Gene expression related to the differentiation to the mesenchymal lineage indicated that CB-MSC can differentiate towards osteoblasts and chondrocytes. CB-MSC derived cell lines maintained normal karyotypes when the cells were cultured up to 40 PDL. Conclusions Among several factors possibly responsible for success in isolating CB-MSC, time between delivery and processing was decisive and volume was also critical. Even though the frequency of CB-MSC was much lower initially than BM-MSC, the high proliferation rate of these cells should allow expansion to cell numbers adequate for clinical use. High proliferation rate combined with high differentiation capability and the karyotype stability after long culture, indicate that CB-MSC should be a potential practical source of MSC for regenerative medicine. Disclosures No relevant conflicts of interest to declare.

scholarly journals Intrapancreatic MSC transplantation facilitates pancreatic islet regeneration

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rahul Khatri ◽  
Sebastian Friedrich Petry ◽  
Thomas Linn
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Pancreatic Islet ◽  
Nude Mice ◽  
Direct Contact ◽  
Islet Cells ◽  
Physical Contact ◽  
Min6 Cells

Abstract Background Type 1 diabetes mellitus (T1D) is characterized by the autoimmune destruction of the pancreatic β cells. The transplantation of mesenchymal stromal/stem cells (MSC) was reported to rescue the damaged pancreatic niche. However, there is an ongoing discussion on whether direct physical contact between MSC and pancreatic islets results in a superior outcome as opposed to indirect effects of soluble factors released from the MSC entrapped in the lung microvasculature after systemic administration. Hence, MSC were studied in direct contact (DC) and indirect contact (IDC) with murine pancreatic β cell line MIN6-cells damaged by nitrosourea derivative streptozotocin (STZ) in vitro. Further, the protective and antidiabetic outcome of MSC transplantation was evaluated through the intrapancreatic route (IPR) and intravenous route (IVR) in STZ-induced diabetic NMRI nude mice. Methods MSC were investigated in culture with STZ-damaged MIN6-cells, either under direct contact (DC) or separated through a semi-permeable membrane (IDC). Moreover, multiple low doses of STZ were administered to NMRI nude mice for the induction of hyperglycemia. 0.5 × 106 adipose-derived mesenchymal stem cells (ADMSC) were transferred through direct injection into the pancreas (IPR) or the tail vein (IVR), respectively. Bromodeoxyuridine (BrdU) was injected for the detection of proliferating islet cells in vivo, and real-time polymerase chain reaction (RT-PCR) was employed for the measurement of the expression of growth factor and immunomodulatory genes in the murine pancreas and human MSC. Phosphorylation of AKT and ERK was analyzed with Western blotting. Results The administration of MSC through IPR ameliorated hyperglycemia in contrast to IVR, STZ, and non-diabetic control in a 30-day window. IPR resulted in a higher number of replicating islet cells, number of islets, islet area, growth factor (EGF), and balancing of the Th1/Th2 response in vivo. Physical contact also provided a superior protection to MIN6-cells from STZ through the AKT and ERK pathway in vitro in comparison with IDC. Conclusion Our study suggests that the physical contact between MSC and pancreatic islet cells is required to fully unfold their protective potential.

scholarly journals Study of Mesenchymal Stem Cells Cultured on a Poly(Lactic-co-Glycolic Acid) Scaffold Containing Simvastatin for Bone Healing

2017 ◽  
Vol 15 (2) ◽  
pp. 133-141 ◽  
Author(s):  
Dario Mendes Junior ◽  
Juliana A. Domingues ◽  
Moema A. Hausen ◽  
Silvia M.M. Cattani ◽  
Aguedo Aragones ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Glycolic Acid ◽  
Bone Growth ◽  
Bone Substitutes ◽  
Tissue Growth ◽  
Main Hypothesis ◽  
Promising Alternative

Background Tissue engineering is a promising alternative for the development of bone substitutes; for this purpose, three things are necessary: stem cells, a scaffold to allow tissue growth and factors that induce tissue regeneration. Methods To congregate such efforts, we used the bioresorbable and biocompatible polymer poly(lactic-co-glycolic acid) (PLGA) as scaffold. For the osteoinductive factor, we used simvastatin (SIM), a drug with a pleiotropic effect on bone growth. Mesenchymal stem cells (MSCs) were cultured in PLGA containing SIM, and the bone substitute of PLGA/SIM/MSC was grafted into critical defects of rat calvaria. Results The in vitro results showed that SIM directly interfered with the proliferation of MSC promoting cell death, while in the pure PLGA scaffold the MSC grew continuously. Scaffolds were implanted in the calvaria of rats and separated into groups: control (empty defect), PLGA pure, PLGA/SIM, PLGA/MSC and PLGA/SIM/MSC. The increase in bone growth was higher in the PLGA/SIM group. Conclusions We observed no improvement in the growth of bone tissue after implantation of the PLGA/SIM/MSC scaffold. As compared with in vitro results, our main hypothesis is that the microarchitecture of PLGA associated with low SIM release would have created an in vivo microenvironment of concentrated SIM that might have induced MSC death. However, our findings indicate that once implanted, both PLGA/SIM and PLGA/MSC contributed to bone formation. We suggest that strategies to maintain the viability of MSCs after cultivation in PLGA/SIM will contribute to improvement of bone regeneration.

scholarly journals Production of insulin producing cells from cord blood mesenchymal stem cells and their potential in cell therapy

2019 ◽  
Vol 9 (6-s) ◽  
pp. 65-71
Author(s):  
Nihad Elsadig Babiker ◽  
Alsadig Gassoum ◽  
Mohamed Abdelrahman Arbab ◽  
Sawsan Ahmed Hamed ALDeaf ◽  
Imad Fadl-Elmula ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Blood Glucose ◽  
Cord Blood ◽  
High Glucose ◽  
Glucose Concentration ◽  
High Glucose Concentration ◽  
Insulin Producing Cells

Introduction: Mesenchymal stem cells (MSCs) were described as adherent cells with a fibroblast-like appearance, have a great capacity for self-renewal while maintaining their multipotency and differentiation into multiple tissues in vivo and in vitro. Methods: MSCs were isolated from cord blood of Sudanese donors using Ficoll-Hypaque gradient density protocol, and differentiate into β- like cells using 3-step protocol. STZ induced diabetic rats were injected intraperitoneally with the differentiated islet β- like cells and blood glucose levels were monitored for seven days. Results: The adherent cell appeared round and sphere after one-week of incubation, and the fibroblast-like colony was strongly attached after three weeks of seeding. The phenotyping of cells showed positivity for CD13, and negativity for CD34, CD45 and HLADR. MSCs were induced into islet-like cells using a 3-step (15-days) protocol. The differentiated cells showed positive diathizone stain and positive imuno-reactivity to anti-human insulin antibody. Secretion of insulin by insulin-producing cells showed positive result with >3.4 u/ml scale reading in high glucose concentration medium. After one-week of transplantation the level of blood glucose was reduced from 410 to 225 mg/dl in the experimental rat. Conclusion: Human UCB-MSCs can be differentiated into insulin-secreting cells invitro, and are able to produce and secrete insulin in response to high glucose concentration in vivo and in vitro. Keywords: Cord blood, Mesenchymal stem cell, islets β-like cells

scholarly journals Quantum Dots Do Not Alter the Differentiation Potential of Pancreatic Stem Cells and Are Distributed Randomly among Daughter Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
S. Danner ◽  
H. Benzin ◽  
T. Vollbrandt ◽  
J. Oder ◽  
A. Richter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Quantum Dots ◽  
Animal Models ◽  
Adult Stem Cells ◽  
Cell Labeling ◽  
Differentiation Potential ◽  
Daughter Cells ◽  
Pancreatic Stem Cells

With the increasing relevance of cell-based therapies, there is a demand for cell-labeling techniques forin vitroandin vivostudies. For the reasonable tracking of transplanted stem cells in animal models, the usage of quantum dots (QDs) for sensitive cellular imaging has major advances. QDs could be delivered to the cytoplasm of the cells providing intense and stable fluorescence. Although QDs are emerging as favourable nanoparticles for bioimaging, substantial investigations are still required to consider their application for adult stem cells. Therefore, rat pancreatic stem cells (PSCs) were labeled with different concentrations of CdSe quantum dots (Qtracker 605 nanocrystals). The QD labeled PSCs showed normal proliferation and their usual spontaneous differentiation potentialin vitro. The labeling of the cell population was concentration dependent, with increasing cell load from 5 nM QDs to 20 nM QDs. With time-lapse microscopy, we observed that the transmission of the QD particles during cell divisions was random, appearing as equal or unequal transmission to daughter cells. We report here that QDs offered an efficient and nontoxic way to label pancreatic stem cells without genetic modifications. In summary, QD nanocrystals are a promising tool for stem cell labeling and facilitate tracking of transplanted cells in animal models.

In Vitro and in Vivo Comparison of Binding of 99m-Tc-Iabeled Anti-CEA MAb F33-104 with 99m-Tc-labeled Anti-CEA MAb BW431/26

1999 ◽  
Vol 38 (04) ◽  
pp. 115-119
Author(s):  
N. Oriuchi ◽  
S. Sugiyama ◽  
M. Kuroki ◽  
Y. Matsuoka ◽  
S. Tanada ◽  
...  
Keyword(s):  
Nude Mice ◽  
Binding Capacity ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Cell Binding ◽  
Vitro Binding ◽  
Labeling Method ◽  
Human Colon Adenocarcinoma

Summary Aim: The purpose of this study was to assess the potential for radioimmunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. Methods: The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. Results: The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tclabeled anti-CEA MAb BW431/261 (31.4 ± 0.95% vs. 11.9 ± 0.55% at 100 ng/mL of soluble CEA, 83.5 ± 2.84% vs. 54.0 ± 2.54% at 107 of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 ± 3.50% ID/g vs. 14.4 ± 3.30% ID/g). 99m-Tcactivity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 ± 2.10% ID/g vs. 8.01 ± 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 ± 1.70% ID/g vs. 8.10 ± 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). Conclusion: 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Significance of In-Vitro and In-Vivo Correlation in Drug Delivery System

2018 ◽  
Vol 4 (4) ◽  
pp. 523-531
Author(s):  
Hina Mumtaz ◽  
Muhammad Asim Farooq ◽  
Zainab Batool ◽  
Anam Ahsan ◽  
Ashikujaman Syed
Keyword(s):  
Dosage Form ◽  
Pharmaceutical Preparations ◽  
Pharmacokinetic Profile ◽  
In Vitro Dissolution ◽  
Time Saving ◽  
Pharmaceutical Dosage Form ◽  
Short Period ◽  
Form Development

The main purpose of development pharmaceutical dosage form is to find out the in vivo and in vitro behavior of dosage form. This challenge is overcome by implementation of in-vivo and in-vitro correlation. Application of this technique is economical and time saving in dosage form development. It shortens the period of development dosage form as well as improves product quality. IVIVC reduce the experimental study on human because IVIVC involves the in vivo relevant media utilization in vitro specifications. The key goal of IVIVC is to serve as alternate for in vivo bioavailability studies and serve as justification for bio waivers. IVIVC follows the specifications and relevant quality control parameters that lead to improvement in pharmaceutical dosage form development in short period of time. Recently in-vivo in-vitro correlation (IVIVC) has found application to predict the pharmacokinetic behaviour of pharmaceutical preparations. It has emerged as a reliable tool to find the mode of absorption of several dosage forms. It is used to correlate the in-vitro dissolution with in vivo pharmacokinetic profile. IVIVC made use to predict the bioavailability of the drug of particular dosage form. IVIVC is satisfactory for the therapeutic release profile specifications of the formulation. IVIVC model has capability to predict plasma drug concentration from in vitro dissolution media.

Autologous transplantation of mesenchymal stem cells into the portal vein of the miniature pig; a preliminary experiment for NOTES approach

2019 ◽  
Vol 98 (9) ◽  
pp. 350-355
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Therapy ◽  
Portal Vein ◽  
Liver Parenchyma ◽  
Miniature Pig ◽  
Hepatic Diseases ◽  
Study Results

Introduction: There is evidence that mesenchymal stem cells (MSCs) could trans-differentiate into the liver cells in vitro and in vivo and thus may be used as an unfailing source for stem cell therapy of liver disease. Combination of MSCs (with or without their differentiation in vitro) and minimally invasive procedures as laparoscopy or Natural Orifice Transluminal Endoscopic Surgery (NOTES) represents a chance for many patients waiting for liver transplantation in vain. Methods: Over 30 millions of autologous MSCs at passage 3 were transplanted via the portal vein in an eight months old miniature pig. The deposition of transplanted cells in liver parenchyma was evaluated histologically and the trans-differential potential of CM-DiI labeled cells was assessed by expression of pig albumin using immunofluorescence. Results: Three weeks after transplantation we detected the labeled cells (solitary, small clusters) in all 10 samples (2 samples from each lobe) but no diffuse distribution in the samples. The localization of CM-DiI+ cells was predominantly observed around the portal triads. We also detected the localization of albumin signal in CM-DiI labeled cells. Conclusion: The study results showed that the autologous MSCs (without additional hepatic differentiation in vitro) transplantation through the portal vein led to successful infiltration of intact miniature pig liver parenchyma with detectable in vivo trans-differentiation. NOTES as well as other newly developed surgical approaches in combination with cell therapy seem to be very promising for the treatment of hepatic diseases in near future.

Differentiation of mesenchymal stem cells from humans and animals into insulin-producing cells: An overview in vitro induction forms

2020 ◽  
Vol 16 ◽  
Author(s):  
Bruna O. S. Câmara ◽  
Bruno M. Bertassoli ◽  
Natália M. Ocarino ◽  
Rogéria Serakides
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture Medium ◽  
Adipogenic Differentiation ◽  
Medium Composition ◽  
Cell Therapies ◽  
Insulin Producing Cells ◽  
Msc Differentiation

The use of stem cells in cell therapies has shown promising results in the treatment of several diseases, including diabetes mellitus, in both humans and animals. Mesenchymal stem cells (MSCs) can be isolated from various locations, including bone marrow, adipose tissues, synovia, muscles, dental pulp, umbilical cords, and the placenta. In vitro, by manipulating the composition of the culture medium or transfection, MSCs can differentiate into several cell lineages, including insulin-producing cells (IPCs). Unlike osteogenic, chondrogenic, and adipogenic differentiation, for which the culture medium and time are similar between studies, studies involving the induction of MSC differentiation in IPCs differ greatly. This divergence is usually evident in relation to the differentiation technique used, the composition of the culture medium, the cultivation time, which can vary from a few hours to several months, and the number of steps to complete differentiation. However, although there is no “gold standard” differentiation medium composition, most prominent studies mention the use of nicotinamide, exedin-4, ß-mercaptoethanol, fibroblast growth factor b (FGFb), and glucose in the culture medium to promote the differentiation of MSCs into IPCs. Therefore, the purpose of this review is to investigate the stages of MSC differentiation into IPCs both in vivo and in vitro, as well as address differentiation techniques and molecular actions and mechanisms by which some substances, such as nicotinamide, exedin-4, ßmercaptoethanol, FGFb, and glucose, participate in the differentiation process.

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