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Posts Tagged ‘FACS’


Reporter: Ritu Saxena, Ph.D.

Diabetes currently affects more than 336 million people worldwide, with healthcare costs by diabetes and its complications of up to $612 million per day in the US alone.  The islets of Langerhans, miniature endocrine organs within the pancreas, are essential regulators of blood glucose homeostasis and play a key role in the pathogenesis of diabetes.  Islets of Langerhans are composed of several types of endocrine cells.  The α- and β-cells are the most abundant and also the most important in that they secrete hormones (glucagon and insulin, respectively) crucial for glucose homeostasis (Bosco D, et al, Diabetes, May 2010;59(5):1202-10).

Diabetes is a ‘bihormonal’ disease, involving both insulin deficiency and excess glucagon.  For decades, insulin deficiency was considered to be the sole reason for diabetes; however, recent studies emphasize excess glucagon as an important part of diabetes etiology.  Thus, insulin-secreting β cells and glucagon-secreting α cells maintain physiological blood glucose levels, and their malfunction drives diabetes development.  Increasing the number of insulin-producing β cells while decreasing the number of glucagon-producing α cells, either in vitro in donor pancreatic islets before transplantation into type 1 diabetics or in vivo in type 2 diabetics, is a promising therapeutic avenue.  A huge leap has been taken in this direction by the researchers at the University of Pennsylvania (Philadelphia, PA) in collaboration with Oregon Health and Science University (Portland, OR), USA by demonstrating that α to β cell reprogramming could be promoted by manipulating the histone methylation signature of human pancreatic islets.  In fact, the treatment of cultured pancreatic islets with a histone methyltransferase inhibitor leads to colocalization of both glucagon and insulin and glucagon and insulin promoter factor 1 (PDX1) in human islets and colocalization of both glucagon and insulin in mouse islets.  The research findings were published in the Journal of Clinical Investigation.

Study design: First step was to study and analyze the epigenetic and transcriptional landscape of human pancreatic human pancreatic α, β, and exocrine cells using ChIP and RNA sequencing.  Study design for determination of the transcriptome and differential histone marks included the dispersion and FACS to of human islets to obtain cell populations highly enriched for α, β, and exocrine (duct and acinar) cells.  Then, chromatin was prepared for ChIP analysis using antibodies for histone modifications, H3K4me3 (represents gene activation) and H3K27me3 (represents gene repression).  RNA-Sequencing analysis was then performed to determine mRNA and lncRNA.  Sample purity was confirmed using qRT-PCR of insulin and glucagon expression levels of the individual α and β cell population revealing high sample purity.

Results:

  • Long noncoding transcripts: Long noncoding RNA molecules have been implicated as important developmental regulators, cell lineage allocators, and contributors to disease development.  The authors discovered 12 cell–specific and 5 α cell–specific noncoding (lnc) transcripts, indicative of the valuable research resource represented from transcriptome data.  Recently discovered lncRNA molecules in islets are regulated during development and dysregulated in type 2 diabetic islets.
  • Monovalent histone modification landscapes shared among three cell types:  Monovalent H3K4me3-enriched regions, indicative of gene activation, were identified and compared in α, β, and exocrine cells.  Strikingly, the vast majority of monovalently H3K4me3-marked genes were shared among the 3 pancreatic cell lineages (83%–95%), reflecting both their related function in protein secretion and common embryonic descent. Similarly, a high degree of overlap was observed in H3K27me3 modification patterns in all the three cell types (73%–83%).
  • Bivalent histone modifications (H3K4me3 and H3K27me3) were high in α cells: Bernstein colleagues observed bivalent marks to be common in undifferentiated cells, such as ES cells and pluripotent progenitor cells, and in most cases, one of the histone modification marks was lost during differentiation, accompanying lineage specification (Bernstein BE, et al, Cell, 21 Apr 2006; 125(2):315-26).  α cells exhibited many more genes bivalently marked, followed by β cells and exocrine cells.  Bivalent state was remarkably similar to that of hESC, suggesting a more plastic epigenomic state for α cells.
  • Monovalent histone modifications were high in β cells: Thousands of the genes that were in bivalent state in α cells were in a monovalent state, carrying only the activating or repressing mark.
  • Inhibition of histone methyltransferases led to partial cell-fate conversion: Adenosine dialdehye (Adox), a drug that interferes with histone methylation and decreases H3K27me3, when administered in human islet tissue, led to decrease of H3K27me3 enrichment at the 3 gene loci that are originally expressed bivalently in α cells and monovalently in β cells:  MAFA, PDX1 and ARX.  Adox resulted in the occasional cooccurrence of glucagon and insulin granules within the same islet cell, which was not observed in untreated islets.  Thus, inhibition of histone methyltransferases leads to partial endocrine cell-fate conversion.

Conclusion:  α cells have been reprogrammed into β cell fate in various mouse models.  The reason, as proposed by the authors, might be the presence of more bivalently marked genes that confers a more plastic epigenomic state of the cells that probably drives them to the β cell fate.  Therefore, using epigenomic information of different cell types in pancreatic islets and harnessing it for subsequent manipulation of their epigenetic signature could be utilized to reprogram cells and hence provide a path for diabetes therapy.

Source: Bramswig NC, et al, Epigenomic plasticity enables human pancreatic α to β cell reprogramming. J Clin Invest, 22 Feb 2013. pii: 66514.

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Junk DNA codes for valuable miRNAs: non-coding DNA controls Diabetes

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Reprogramming cell fate

CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC

2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single Human Cell

SNAP: Predict Effect of Non-synonymous Polymorphisms: How well Genome Interpretation Tools could Translate to the Clinic

Genomic Endocrinology and its Future

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Author and Reporter: Ritu Saxena, Ph.D.

Introduction

Blood vessels arise from endothelial precursors that are thin, flat cells lining the inside of blood vessels forming a monolayer throughout the circulatory system. ECs are defined by specific cell surface markers including CD31, CD34, CD105, VE-cadherin, vascular endothelial growth factor receptor 1 [VEGFR-1], VEGFR-2, Tie-1, Tie-2) that characterize their phenotype. Angiogenesis is the growth of new blood vessels from preexisting ones and is required for growth and repair. Malignancy is a pathological scenario that requires angiogenesis. The definite cellular origin of adult blood vessel-forming cells necessary for neoangiogenesis has been unknown. Weissman and fellow coworkers in their previous work indicated that the address of these cells might be local, residing in non-circulating tissue. Also, very low numbers of cells with endothelial characteristics and high proliferative potential have been reported in umbilical cord blood or in peripheral blood. The function of circulating endothelial progenitor cells and pharmacotherapy targeted at the      endogenous augmentation of these cells for their use in cardiovascular repair has been discussed in detail in a post authored by Aviva Lev-Ari on August 28, 2012.

Research

Scientists at the University of Helsinki, Finland, wanted to find out if there exists a rare vascular endothelial stem cell (VESC) population that is capable of producing very high numbers of endothelial daughter cells, and can lead to neovascular growth in adults.  They were not only able to define the characteristic cells responsible for giving rise of blood vessels in adults, but took a leap forward by generating blood vessels from a single cells from the VESC population. (Figure:  VESCs discovered that reside at the blood vessel wall endothelium. These are a small population of CD117+ ECs capable of self-renewal.  Image Courtesy: Fang et al, 2012).

The VESCs, as explained by the Fang and coworkers, reside in the blood vessel wall endothelium and constitute a small subpopulation within CD117+ (c-kit+) endothelial cells (ECs). These cells are capable of undergoing clonal expansion unlike the surrounding ECs that bear limited proliferating potential. VESC discovered in this study were found to a have a certain characteristic phenotype defined by the presence of a few surface proteins. The authors utilized the technique of FACS (Fluorescence Activated Cell Sorting) to isolate the cells capable of undergoing clonal expansion. The sorting was performed against endothelial-specific protein markers CD31 and CD15, and against CD117 and Sca-1 molecules that are expressed by many adult stem cell types including hematopoietic stem cells (HSCs) and prostate and mammary gland stem cells. The experimental results defined the surface characteristics or the phenotype of the isolated cells to be lin2CD31+CD105+Sca1+CD117+A.  A single VESC cell isolated from the endothelial population was able to generate functional blood vessels that connected to host circulation after transplantation in mouse. In cell culture, these cells were shown to generate tens of millions of daughter endothelial cells. Also, within cell culture, the isolated VESCs showed long-term self-renewal properties, bearing similarity to adult stem cells. The self-renewal capacity of VESCs was evident even in vivo, when the ‘isolated’ ECs containing VESCs retained the capacity to generate functional blood vessels during serial transplantations. The transplanted ECs were monitored with the help of Green Fluorescent protein (GFP). Fluorescent blood vessels were observed in secondary, tertiary, and quaternary transplants providing direct evidence that the GFP-tagged ECs contained VESCs with self-renewal capacity.

Furthermore, the cell culture and animal experiment results were supported by the observation that abundant CD117+ ECs were discovered in human malignant melanomas and invasive breast cancer samples.

Research relevance

The discovery of VESCs is seminal and could be of tremendous therapeutic potential. It could be useful in the following ways leading way for related research endeavors including-

  • Cell-based therapies: VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization i.e., the daughter cells arising from VESCs at the target site could assist in repair by generation of  neoangiogenic ECs required for the formation of blood vessels.
  • Therapeutic target: VESCs could serve as a possible cellular and molecular target to restrain angiogenesis by inhibiting endothelial-cell proliferation thereby blocking cancer progression.

Sources:

Fang S et al, Generation of Functional Blood Vessels from a Single c- kit + Adult Vascular Endothelial Stem Cell. PLoS Biol. 2012;10(10):e1001407. http://www.ncbi.nlm.nih.gov/pubmed/23091420

News Brief: http://www.business-standard.com/generalnews/news/scientists-discover-new-blood-vessel-generating-cells/69329/

Related reading:

Cardiovascular and endothelial cells

Statins’ Nonlipid Effects on Vascular Endothelium through eNOS Activation Curator, Author,Writer, Reporter: Larry Bernstein, MD, FCAP

Cardiovascular Outcomes: Function of circulating Endothelial Progenitor Cells (cEPCs): Exploring Pharmaco-therapy targeted at Endogenous Augmentation of cEPCs Author and Curator: Aviva Lev-Ari, PhD, RN

Vascular Medicine and Biology: Macrovascular Disease – Therapeutic Potential of cEPCs Curator and Author: Aviva Lev-Ari, PhD, RN

Repair damaged blood vessels in heart disease, stroke, diabetes and trauma: Cellular Reprogramming amniotic fluid-derived cells into Endothelial Cells Reporter: Aviva Lev-Ari, PhD, RN

Stem cells in therapy

A possible light by Stem cell therapy in painful dark of Osteoarthritis” – Kartogenin, a small molecule, differentiates stem cells to chondrocyte, healthy cartilage cells Author and Reporter: Anamika Sarkar, Ph.D and Ritu Saxena, Ph.D.

Human embryonic pluripotent stem cells and healing post-myocardial infarction Author: Larry H. Bernstein, MD

Stem cells create new heart cells in baby mice, but not in adults, study shows Reporter: Aviva Lev-Ari, PhD, RN

Stem cells for the rescue of mitochondrial dysfunction in Parkinson’s disease Reporter: Ritu Saxena, Ph.D.

Stem Cell Research — The Frontier is at the Technion in Israel Reporter: Aviva Lev-Ari, PhD, RN

Research articles by MA Gaballa, PhD

Harris DT, Badowski M, Nafees A, Gaballa MA. The potential of Cord Blood Stem Cells for Use in Regenerative Medicine. Expert Opinion in Biological Therapy 2007. Sept 7(9): 1131-22.

Furfaro E, Gaballa MA. Do adult stem cells ameliorate the damaged myocardium?. Human cord blood as a potential source of stem cells. Current Vascular Pharmacology 2007, 5; 27-44.

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