scholarly journals Human fetal pancreatic insulin-producing cells proliferate in vitro

2009 ◽  
Vol 201 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Mugdha V Joglekar ◽  
Vinay M Joglekar ◽  
Sheela V Joglekar ◽  
Anandwardhan A Hardikar
Keyword(s):  
Pancreatic Islets ◽  
Endocrine Cells ◽  
Single Cells ◽  
Mesenchymal Cell ◽  
Cell Populations ◽  
Open Chromatin ◽  
Insulin Producing Cells ◽  
Pancreatic Insulin ◽  
Insulin Promoter

There have been considerable efforts towards understanding the potential of human pancreatic endocrine cells to proliferate and transition into mesenchymal cell populations. Since rodent studies have demonstrated that mouse insulin-producing cells do not proliferate in vitro, a similar possibility has been considered for human islet endocrine cells. Considering the inherent differences in mouse and human pancreatic islets, we decided to assess the potential of human fetal pancreatic insulin-producing cells to proliferate in vitro. We studied the proliferative potential of human fetal pancreatic islet-derived populations from second or third trimester fetal pancreas and characterized the cells that grow out during their expansion. We have used seven different approaches including in situ hybridization and immunostaining, quantitative estimation of multiple gene transcripts in populations as well as in single cells, clonal analysis of islet cells, assessment of heritable marks of active insulin promoter, and thymidine analog-based lineage tracing. Our studies demonstrate that human fetal pancreatic insulin-producing cells proliferate in vitro to generate mesenchymal cell populations. Interestingly, epigenetic modifications that mark open chromatin conformation of insulin promoter regions are retained even after a million fold expansion/proliferation in vitro. These findings demonstrate that hormone-producing cells in pancreatic islets proliferate in vitro and retain epigenetic marks that characterize an active insulin promoter. Such in vitro-derived mesenchymal cells may be of potential use in cell-replacement therapy for diabetes.

Isolation of the Stromal/Vascular Fraction of Murine Bone Marrow Dramatically Enhances MSC Yield, Allows Isolation of Marrow Endothelial Cells and Reveals Multiple Subpopulations of Stromal Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3861-3861
Author(s):  
Colby G. Suire ◽  
Nathalie Brouard ◽  
Brian Blaugrund ◽  
Paul J. Simmons
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Single Cells ◽  
Cell Populations ◽  
Multilineage Differentiation ◽  
Differentiation Potential ◽  
Cellular Therapies

Abstract Abstract 3861 The bone marrow is the organ of residence of a population of multipotent progenitor cells most commonly referred to as mesenchymal stem cells (MSC) based upon their multilineage differentiation potential into bone, cartilage and adipose tissue. The capacity for MSC to contribute to tissue repair demonstrated by numerous previous reports has engendered considerable interest in their application to a broad range of cellular therapies. It follows that a robust reproducible methodology for obtaining high yields of MSC from preclinical animal models, such as rodents, would greatly facilitate the development of these various MSC-based cellular therapies. The plastic-adherent, clonogenic progenitors termed colony forming unit-fibroblast (CFU-F) originally identified by Freidenstein and colleagues that initiate MSC cultures are a rare population in the marrow of all mammalian species so far examined. This is particularly so in the case of the mouse where reported incidences of CFU-F are typically in the range of 1/200,000 bone marrow BM cells. The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of MSC from mouse BM, a common problem being contamination with hematopoietic cells. Seeking to develop an improved methodology to harvest MSC from mouse using methods based on plastic adherent bone marrow, we took advantage of burgeoning evidence demonstrating the perivascular location of MSC not only in the bone marrow, but also in multiple tissues. We hypothesized that a potential reason for the low yield of mMSC from mBM is the flushing of the marrow used to remove single cells suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of MSC physically associated with the abluminal surface of blood vessels. Herein, we describe a simple methodology based on preparation of intact marrow plugs that yields distinct populations of both stromal and endothelial cells. BM plugs are subjected to 3 sequential rounds of digestion in collagenase/dispase and each fraction assayed for content of CFU-F. The recovery of CFU-F obtained by pooling the product of each digestion (1643+199) reproducibly exceeds that obtained using the standard BM flushing technique (13.3+1.9) by at least 2 orders of magnitude (P=<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal cell populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and these demonstrate robust multilineage differentiation into bone, adipose and chondrogenic progeny using standard in vitro bioassays. A detailed immunophenotypic analysis of the P0 cultures demonstrated the existence of multiple stromal cell subpopulations many of the markers analyzed, including Sca-1, CD90, CD105, CD146 and PDGFRa, which was progressively lost with serial passaging. Discrete subpopulations of stromal cells identified at P0, in many cases had phenotypically identical counterparts in the BM cell suspensions prepared by serial digestion and we are in the process of quantitatively analyzing the evolution of selected phenotypes in vitro to provide clues as to the identity of the founder population of stromal cells that gives rise to ‘MSC' in vitro. Finally, the phenotypic analysis of P0 cultures also revealed a discrete population of CD105BrightPDGFRaNeg cells representing a mean of 26.7% of hematopoietic lineage-negative cells. Upon isolation and serial propagation, the cells maintain expression of all of the vascular endothelial markers examined including CD31, CD105, VCAM-1, CD144 and MECA32 and also demonstrate inducible expression of E-selectin upon treatment with TNF-a. In conclusion, we describe a simple and robust methodology that, for the first time, allows the simultaneous isolation of both the stromal and vascular components of mouse BM. Secondly, the yield of ‘MSC' afforded by this technique far exceeds that reported in any previous study. Thirdly, this technique reveals a level of stromal cell heterogeneity not apparent in previous analyses of mouse BM-derived MSC that more realistically reflects the likely complexity of stromal cell populations in vivo and represents a platform for the eventual prospective isolation of specific subpopulations. These studies will greatly enhance experimental strategies designed to analyze not only MSC identity but also the function of the vascular hematopoietic niche. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Activin A, exendin-4, and glucose stimulate differentiation of human pancreatic ductal cells

2013 ◽  
Vol 217 (3) ◽  
pp. 241-252 ◽  
Author(s):  
Hyo-Sup Kim ◽  
Seung-Hyun Hong ◽  
Seung-Hoon Oh ◽  
Jae-Hyeon Kim ◽  
Myung-Shik Lee ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Pancreatic Islets ◽  
Nude Mice ◽  
High Glucose ◽  
Activin A ◽  
Alternative Source ◽  
Ductal Cells ◽  
Pancreatic Ductal Cells ◽  
Insulin Producing Cells

Islet transplantation is one treatment option for diabetes mellitus. However, novel sources of pancreatic islets or insulin-producing cells are required because the amount of donor tissue available is severely limited. Pancreatic ductal cells are an alternative source of β-cells because they have the potential to differentiate into insulin-producing cells. We investigated whether treatment of human pancreatic ductal cells with activin A (ActA) and exendin-4 (EX-4) stimulated transdifferentiation of the cells, bothin vitroandin vivo. We treated human pancreatic ductal cells with ActA and EX-4 in high-glucose media to induce differentiation into insulin-producing cells and transplanted the cells into streptozotocin-induced diabetic nude mice. Co-treatment of mice with ActA and EX-4 promoted cell proliferation, induced expression of pancreatic β-cell-specific markers, and caused glucose-induced insulin secretion compared with the ActA or EX-4 mono-treatment groups respectively. When pancreatic ductal cells treated with ActA and EX-4 in high-glucose media were transplanted into diabetic nude mice, their blood glucose levels normalized and insulin was detected in the graft. These findings suggest that pancreatic ductal cells have a potential to replace pancreatic islets for the treatment of diabetes mellitus when the ductal cells are co-treated with ActA, EX-4, and glucose to promote their differentiation into functional insulin-producing cells.

scholarly journals Single cell ATAC-seq in human pancreatic islets and deep learning upscaling of rare cells reveals cell-specific type 2 diabetes regulatory signatures

2019 ◽  
Author(s):  
Vivek Rai ◽  
Daniel X. Quang ◽  
Michael R. Erdos ◽  
Darren A. Cusanovich ◽  
Riza M. Daza ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Deep Learning ◽  
Single Cell ◽  
Pancreatic Islets ◽  
Cell Populations ◽  
Open Chromatin ◽  
Cell Type ◽  
Genome Wide ◽  
Delta Cell

ABSTRACTObjectiveType 2 diabetes (T2D) is a complex disease characterized by pancreatic islet dysfunction, insulin resistance, and disruption of blood glucose levels. Genome wide association studies (GWAS) have identified >400 independent signals that encode genetic predisposition. More than 90% of the associated single nucleotide polymorphisms (SNPs) localize to non-coding regions and are enriched in chromatin-defined islet enhancer elements, indicating a strong transcriptional regulatory component to disease susceptibility. Pancreatic islets are a mixture of cell types that express distinct hormonal programs, and so each cell type may contribute differentially to the underlying regulatory processes that modulate T2D-associated transcriptional circuits. Existing chromatin profiling methods such as ATAC-seq and DNase-seq, applied to islets in bulk, produce aggregate profiles that mask important cellular and regulatory heterogeneity.MethodsWe present genome-wide single cell chromatin accessibility profiles in >1,600 cells derived from a human pancreatic islet sample using single-cell-combinatorial-indexing ATAC-seq (sci-ATAC-seq). We also developed a deep learning model based on the U-Net architecture to accurately predict open chromatin peak calls in rare cell populations.ResultsWe show that sci-ATAC-seq profiles allow us to deconvolve alpha, beta, and delta cell populations and identify cell-type-specific regulatory signatures underlying T2D. Particularly, we find that T2D GWAS SNPs are significantly enriched in beta cell-specific and cross cell-type shared islet open chromatin, but not in alpha or delta cell-specific open chromatin. We also demonstrate, using less abundant delta cells, that deep-learning models can improve signal recovery and feature reconstruction of rarer cell populations. Finally, we use co-accessibility measures to nominate the cell-specific target genes at 104 non-coding T2D GWAS signals.ConclusionsCollectively, we identify the islet cell type of action across genetic signals of T2D predisposition and provide higher-resolution mechanistic insights into genetically encoded risk pathways.

scholarly journals Mesenchymal Cell Community Effect in Whole Tooth Bioengineering

2016 ◽  
Vol 96 (2) ◽  
pp. 186-191 ◽  
Author(s):  
L. Yang ◽  
A. Angelova Volponi ◽  
Y. Pang ◽  
P.T. Sharpe
Keyword(s):  
Cell Expansion ◽  
Mesenchymal Cell ◽  
Tooth Pulp ◽  
Cell Populations ◽  
Embryonic Cells ◽  
Tooth Formation ◽  
Bioengineered Tooth ◽  
Pulp Cells ◽  
Mesenchyme Cells

In vitro expanded cell populations can contribute to bioengineered tooth formation but only as cells that respond to tooth-inductive signals. Since the success of whole tooth bioengineering is predicated on the availability of large numbers of cells, in vitro cell expansion of tooth-inducing cell populations is an essential requirement for further development of this approach. We set out to investigate if the failure of cultured mesenchyme cells to form bioengineered teeth might be rescued by the presence of uncultured cells. To test this, we deployed a cell-mixing approach to evaluate the contributions of cell populations to bioengineered tooth formation. Using genetically labeled cells, we are able to identify the formation of tooth pulp cells and odontoblasts in bioengineered teeth. We show that although cultured embryonic dental mesenchyme cells are unable to induce tooth formation, they can contribute to tooth induction and formation if combined with noncultured cells. Moreover, we show that teeth can form from cell mixtures that include embryonic cells and populations of postnatal dental pulp cells; however, these cells are unable to contribute to the formation of pulp cells or odontoblasts, and at ratios of 1:1, they inhibit tooth formation. These results indicate that although in vitro cell expansion of embryonic tooth mesenchymal cells renders them unable to induce tooth formation, they do not lose their ability to contribute to tooth formation and differentiate into odontoblasts. Postnatal pulp cells, however, lose all tooth-inducing and tooth-forming capacity following in vitro expansion, and at ratios >1:3 postnatal:embryonic cells, they inhibit the ability of embryonic dental mesenchyme cells to induce tooth formation.

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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