Over the past few years, extensive efforts have been made to generate in-vitro pancreatic micro-tissue
,
for disease modeling or cell replacement approaches in pancreatic related diseases such as ...diabetes mellitus. To obtain these goals, a closer look at the diverse cells participating in pancreatic development is necessary. Five major non-epithelial pancreatic (pN-Epi) cell populations namely, pancreatic endothelium, mesothelium, neural crests, pericytes, and stellate cells exist in pancreas throughout its development, and they are hypothesized to be endogenous inducers of the development. In this review, we discuss different pN-Epi cells migrating to and existing within the pancreas and their diverse effects on pancreatic epithelium during organ development mediated via associated signaling pathways, soluble factors or mechanical cell–cell interactions. In-vivo and in-vitro experiments, with a focus on N-Epi cells’ impact on pancreas endocrine development, have also been considered. Pluripotent stem cell technology and multicellular three-dimensional organoids as new approaches to generate pancreatic micro-tissues have also been discussed. Main challenges for reaching a detailed understanding of the role of pN-Epi cells in pancreas development in utilizing for in-vitro recapitulation have been summarized. Finally, various novel and innovative large-scale bioengineering approaches which may help to recapitulate cell–cell interactions and are crucial for generation of large-scale in-vitro multicellular pancreatic micro-tissues, are discussed.
Adipose tissue-derived mesenchymal stem cells (AD-MSCs) are an appropriate source for regenerative medicine and transplantation therapy due to their high proliferation rate in vitro. ...Bromodeoxyuridine (BrdU) marker is widely used for evaluating cell proliferation. BrdU cell labeling method has some limitations including its effects on cell proliferation and cellular mutations followed by an abnormality in the cell. On the other hand, the city of Kiel-67 (Ki-67) nuclear protein is expressed in dividing cells throughout the mitotic process. Our main objective was to compare the effects of BrdU and Ki-67 markers on self-renewal levels in AD-MSCs. In this study, the AD-MSCs were extracted from subcutaneous adipose tissue of adult rats and cultured in α-MEM supplemented with 10% fetal bovine serum (FBS). Fourth passage AD-MSCs after characterization were evaluated by immunocytochemical assay for BrdU and Ki-67 markers based on growth and proliferation rates of AD-MSCs at 24, 48, 72, and 96 h after culture. Our results show that the growth rate of AD-MSCs significantly changed at different times for both markers, although the growth and proliferation patterns were initially different in them. Unlike the BrdU, the Ki-67 endogenous marker had no side effect and interfered with cellular proliferation. It is opined that evaluating cell proliferation rate using the Ki-67 marker is more effective than the BrdU assay.
Efficient production of functional and mature alveolar epithelial is a major challenge for developing any cell replacement therapy for lung degenerative diseases. The extracellular matrix (ECM) ...pro-vides a dynamic environment and mediates cellular responses during development and maintenance of tissue functions. The decellularized ECM (dECM) which retains its native-like structure and bio-chemical composition can provide the induction of embryonic stem cell (ESC) differentiation toward the tissue-specific lineages during
culture. Therefore, the aim of this study was to evaluate the effect of sheep lung dECM-derived scaffold on differentiation and further maturation of ESC-derived lung progenitor cells.
This study was an experimental study. In the first step, a sheep lung was decellularized to achieve dECM scaffolds and hydrogels. Afterwards, the obtained dECM scaffold was evaluated for collagen and glycosaminoglycan contents, DNA quantification, and its ultrastructure. Next, the three experimental groups: i. Sheep lung dECM-derived scaffold, ii. Sheep lung dECM-derived hydrogel, and iii. Fibronectin-coated plates were compared in their abilities to induce further differentiation of human embryonic stem cells (hESCs)-derived definitive endoderm (DE) into lung progenitor cells. The comparison was evaluated by immuno-staining and real-time polymerase chain reaction (PCR) assessments.
We found that the dECM-derived scaffold preserved its composition and native porous structures while lacking nuclei and intact cells. All experimental groups displayed lung progenitor cell differen-tiation as revealed by the RNA and protein expression of NKX2.1, P63 and CK5. DE cells differenti-ated on dECM-derived scaffold and dECMderived hydrogel showed significant upregulation of
gene expression, a marker of the distal airway epithelium. DE cells differentiated on the dECM-derived scaffold compared to the two other groups, showed enhanced expression of
(type 2 alveolar epithelial AT2 cell marker),
(ciliated cell marker), and
(secretory cell marker) genes.
Overall, our results suggest that dECM-derived scaffold improves the differentiation of DE cells towards lung alveolar progenitor cells in comparison with dECM-derived hydrogel and fibronectin-coated plates.
Growth factors are key elements of embryonic stem cell (ESC) research. Cell line development in eukaryotes is a time-consuming procedure which usually takes 12-18 months. Here, we report an easy and ...fast method with which production of Chinese hamster ovary (CHO) cells that express and secrete recombinant Activin A, as a major growth factor in endo/mesoderm differentiation of embryonic stem cells is achieved within 3-4 weeks.
In this experimental study, we cloned human Activin A into the pDONR/Zeo gateway entry vector using the BP reaction. Activin A was subcloned next into the pLIX_403 and pLenti6.3/TO/V5-DEST destination vectors by the LR reaction. The result was the production of constructs with which 293T cells were finally transfected for virus production. CHO cells were transduced using viral particles to produce a cell line that secretes the His6- Activin A fusion protein.
We developed a quick protocol which saves up to 3-4 weeks of time for producing recombinant proteins in CHO cells. The recombinant cell line produced 90 mg/L of functional Activin A measured in human ESC line Royan H5 (RH5), during in vitro differentiation into meso-endoderm and definitive endoderm.
Our results showed no significant differences in functionality between commercial Activin A and the one produced using our novel protocol. This approach can be easily used for producing recombinant proteins in CHO.
Mesenchymal cells of diverse origins differ in gene and protein expression besides producing varying effects on their organ-matched epithelial cells' maintenance and differentiation capacity. ...Co-culture with rodent's tissue-specific pancreatic mesenchyme accelerates proliferation, self-renewal, and differentiation of pancreatic epithelial progenitors. Therefore, in our study, the impact of three-dimensional (3D) co-culture of human fetal pancreatic-derived mesenchymal cells (hFP-MCs) with human embryonic stem cell-derived pancreatic progenitors (hESC-PPs) development towards endocrine and beta cells was assessed. Besides, the ability to maintain scalable cultures combining hFP-MCs and hESC-PPs was investigated. hFP-MCs expressed many markers in common with bone marrow-derived mesenchymal stem cells (BM-MSCs). However, they showed higher expression of DESMIN compared to BM-MSCs. After co-culture of hESC-PPs with hFP-MCs, the pancreatic progenitor (PP) spheroids generated in Matrigel had higher expression of NGN3 and INSULIN than BM-MSCs co-culture group, which shows an inductive impact of pancreatic mesenchyme on hESC-PPs beta-cells maturation. Pancreatic aggregates generated by forced aggregation through scalable AggreWell system showed similar features compared to the spheroids. These aggregates, a combination of hFP-MCs and hESC-PPs, can be applied as an appropriate tool for assessing endocrine-niche interactions and developmental processes by mimicking the pancreatic tissue.
Organoids can be regarded as a beneficial tool for discovery of new therapeutics for diabetes and/or maturation of pancreatic progenitors (PP) towards β cells. Here, we devised a strategy to enhance ...maturation of PP by assembly of three‐dimensional (3D) pancreatic organoids (PO) containing human embryonic stem (ES) cell derivatives including ES‐derived pancreatic duodenal homeobox 1 (PDX1)
+ early PP, mesenchymal stem cells, and endothelial cells at an optimized cell ratio, on Matrigel. The PO was placed in a 3D‐printed tissue trapper and heterotopically implanted into the peritoneal cavity of immunodeficient mice where it remained for 90 days. Our results indicated that, in contrast to corresponding early PP transplants, 3D PO developed more vascularization as indicated by greater area and number of vessels, a higher number of insulin‐positive cells and improvement of human C‐peptide secretions. Based on our findings, PO‐derived β cells could be considered a novel strategy to study human β‐cell development, novel therapeutics, and regenerative medicine for diabetes.
Organoids can be regarded as a beneficial tool for discovery of new therapeutics for diabetes and/or maturation of pancreatic progenitors (PP) towards β cells. Here, we devised a strategy to enhance maturation of PP by assembly of three‐dimensional (3D) pancreatic organoids (PO) containing human embryonic stem (ES) cell‐derivatives. The PO was placed in a 3D‐printed tissue trapper and heterotopically implanted into the peritoneal cavity of immunodeficient mice where it remained for 90 days. Our results indicate that 3D PO developed more vascularization, a higher number of insulin‐positive cells and improvement of human C‐peptide secretions. ES‐PP: ES cell‐derived pancreatic progenitors; ES‐MSC: ES cell‐derived mesenchymal stem cells; ES‐EC: ES cell‐derived endothelial cells; GF: growth factor; GFR: growth factor reduced; SM: small molecule; IP Tx: intraperitoneal transplantation
•High therapeutic doses of Carbamazepine, Gabapentin, Lamotrigine, Levetiracetam or Topiramate were investigated in an in-vitro study on a human embryonic stem cell line.•Levetiracetam and Topiramate ...were found to damage the DNA significantly compared to untreated cells.•A considerable reduction in DNA damages (genotoxicity) in the presence of Folic acid.
Several antiepileptic drugs (AEDs) are administrated during pregnancy according to recent therapeutic protocols. Ten percent of pregnant women with epilepsy give birth to offspring with malformations and teratogenic defects. Since the mechanism of action of AEDs is not yet completely understood, therefore, it could be hypothesized that they may cause cyto- or genotoxicity in embryonic fetus cells.
To investigate this hypothesis, the genotoxicity and cell survival of AEDs treated human embryonic stem cells (hESCs) were investigated by single-cell gel electrophoresis (Comet assay) and MTS assay, respectively. HESCs (Royan H6 cell line) were treated in-vitro with high therapeutic doses of Carbamazepine, Gabapentin, Lamotrigine, Levetiracetam or Topiramate as monotherapy or combination therapy of each drug with Folic acid.
After hESCs pluripotency confirmation, the effect of AEDs on cellular DNA damage of hESCs was investigated. levetiracetam and topiramate were found to damage the DNA significantly compared to untreated cells. The amount of DNA damage produced by carbamazepine and lamotrigine was similar while for gabapentin, the amount of DNA migration was very low and produced less DNA damage than the others. A considerable reduction in DNA damages occurred in genotoxicity in the presence of Folic acid in comparison to AEDs monotherapies.
According to our results, all mentioned AEDs caused DNA damage, while Levetiracetam and topiramate caused more extensive DNA damages than the others. Noticeably, the addition of Folic acid to the treated cells decreased the DNA damages considerably.
Human pluripotent stem cells (hPSCs) are commonly kept in a primed state but also able to acquire a more immature naive state under specific conditions in vitro. Acquisition of naive state changes ...several properties of hPSCs and might affect their contribution to embryonic development in vivo. However, the lack of an appropriate animal test system has made it difficult to assess potential differences for chimera formation between naive and primed hPSCs. Here, we report that the developing chicken embryo is a permissive host for hPSCs, allowing analysis of the pluripotency potential of hPSCs. Transplantation of naive-like and primed hPSCs at matched developmental stages resulted in robust chimerism. Importantly, the ability of naive-like but not of primed hPSCs to form chimera was substantially reduced when injected at non-matched developmental stages. We propose that contribution to chick embryogenesis is an informative and versatile test to identify different pluripotent states of hPSCs.
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•Developing chicken embryo is a permissive host for hPSCs•Naive and primed hPSCs at matched developmental stages resulted in chicken chimeras•hPSCs differentiate into different cell lineages in xenogeneic chicken chimeras•Chimera reduced in naive hPSCs when injected at non-matched developmental stages
In this article, Hassani, Baharvand, and colleagues show that developmental similarity rather than evolutionary distance in early developmental stages is an important factor in generating human interspecies chimera. Accordingly, chicken embryo provides an applicable system for evaluating naive and primed human pluripotency in stage- and non-stage-matched approaches, albeit with the lower contribution of naive cells in a non-stage-matched manner.
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