Differentiation Of Adult Progenitor Cells Research Articles

5-Hydroxymethylcytosine (5hmC), or How to Identify Your Favorite Cell

Recently described as the sixth base of the DNA macromolecule, the precise role of 5-hydroxymethylcytosine (5hmC) is the subject of debate. Early studies indicate that it is functionally distinct from cytosine DNA methylation (5mC), and there is evidence for 5hmC being a stable derivate of 5mC, rather than just an intermediate of demethylation. Moreover, 5hmC events correlate in time and space with key differentiation steps in mammalian cells. Such events span the three embryonic germ layers and multiple progenitor cell subtypes, suggesting a general mechanism. Because of the growing understanding of the role of progenitor cells in disease origin, we attempted to provide a detailed summary on the currently available literature supporting 5hmC as a key player in adult progenitor cell differentiation. This summary consolidates the emerging role for 5hmC in defining cellular fate.

MR imaging of BioCartilage augmented microfracture surgery utilizing 2D MOCART and KOOS scores

ObjectivesBioCartilage is a novel scaffold-based microfracture augmentation technique that has been shown to aid in chondrogenic differentiation of adult progenitor cells resulting in formation of more hyaline-like cartilage. As this cartilage repair technique becomes more commonplace, it is essential that the musculoskeletal radiologist and orthopedic surgeon gain familiarity with the surgical technique and its post-operative MR imaging findings. MethodsWe present several case studies regarding MRI findings (modified clinical 2D MOCART) and clinical outcome (KOOS) scores in patients who have undergone this novel surgical procedure. For data analysis KOOS scores where dichotomized to scores greater or less than 80, and MOCART scores were dichotomized to scores greater or less than 50. A fisher exact test was then performed to determine if there was any correlation between parameters of the modified 2D MOCART and KOOS scores (Tables 2 and 3). ResultsMarrow elements travel through the microfracture holes and interact with the scaffold created by BioCartilage, rather than creating their own fibrin scaffold, as is typically anticipated from a marrow stimulation procedure. Interestingly, the amount defect fill, presence of an intact surface, intact subchondral bone, or lack of effusion did not correlate with positive outcomes. Parameters that trended towards significance included presence of adhesions and subchondral lamina. Completeness of cartilage interface, homogeneity, and signal intensity also failed to reach statistical significance. In our experience, patients that demonstrated mild repair tissue surface irregularity, but with preservation of greater than 50% thickness compared to surrounding native cartilage, mild irregularity of subchondral plate, with vertical low signal intensity lines (sequela of prior microfracture surgery), and mild or no bone marrow edema pattern demonstrated higher KOOS scores. ConclusionBiocartilage in conjunction with microfracture is an encouraging cartilage restoration technique that promotes regeneration of more robust hyaline-like cartilage compared to the fibrocartilage formed after conventional microfracture. The T2 mapping properties of the repair tissue after successful BioCartilage augmented microfracture surgery are very similar to that of the adjacent native cartilage. Although there appear to be characteristic trends in a successful repair, further research is warranted to elucidate any correlations between specific characteristics of the repair and patient clinical outcomes.

Role of Fibulin 3 in Aging-Related Joint Changes and Osteoarthritis Pathogenesis in Human and Mouse Knee Cartilage.

The EFEMP1 gene encoding fibulin 3 is specifically expressed in the superficial zone (SZ) of articular cartilage. The aims of this study were to examine the expression patterns of fibulin 3 in the knee joints during aging and during osteoarthritis (OA) and to determine the role of fibulin 3 in the pathogenesis of OA. Immunohistochemical analysis was performed on normal and OA knee cartilage samples from humans and mice. Experimental OA was induced in wild-type and fibulin 3-/- mice, and the severity of OA was evaluated by histologic scoring. To examine fibulin 3 function, human chondrocyte monolayer cultures were transfected with small interfering RNA (siRNA), followed by quantitative polymerase chain reaction and Western blot analyses. Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transduced with an EFEMP1 lentivirus and analyzed for markers of chondrogenesis. Fibulin 3 was specifically expressed in the SZ of normal knee joint cartilage from humans and mice, and the expression levels declined with aging. Both aging-related OA and experimental OA were significantly more severe in fibulin 3-/- mice compared with wild-type mice. Fibulin 3 expression was high in undifferentiated human BM-MSCs and decreased during chondrogenesis. Suppression of fibulin 3 by siRNA significantly increased the expression of SOX9, type II collagen, and aggrecan in human articular chondrocytes, while overexpression of fibulin 3 inhibited chondrogenesis in BM-MSCs. Fibulin 3 is specifically expressed in the SZ of articular cartilage and its expression is reduced in aging and OA. Fibulin 3 regulates differentiation of adult progenitor cells, and its aging-related decline is an early event in the pathogenesis of OA. Preventing aging-associated loss of fibulin 3 or restoring it to normal levels in SZ chondrocytes has the potential to delay or prevent the onset of OA.

Differentiation of progenitors in the liver: a matter of local choice.

The liver is a complex organ that requires multiple rounds of cell fate decision for development and homeostasis throughout the lifetime. During the earliest phases of organogenesis, the liver acquires a separate lineage from the pancreas and the intestine, and subsequently, the liver bud must appropriately differentiate to form metabolic hepatocytes and cholangiocytes for proper hepatic physiology. In addition, throughout life, the liver is bombarded with chemical and pathological insults, which require the activation and correct differentiation of adult progenitor cells. This Review seeks to provide an overview of the complex signaling relationships that allow these tightly regulated processes to occur.

DNA methylation restricts spontaneous multi-lineage differentiation of mesenchymal progenitor cells, but is stable during growth factor-induced terminal differentiation

The progressive restriction of differentiation potential from pluripotent embryonic stem cells, via multipotent progenitor cells to terminally differentiated, mature somatic cells, involves step-wise changes in transcription patterns that are tightly controlled by the coordinated action of key transcription factors and changes in epigenetic modifications. While previous studies have demonstrated tissue-specific differences in DNA methylation patterns that might function in lineage restriction, it is unclear at what exact developmental stage these differences arise. Here, we have studied whether terminal, multi-lineage differentiation of C2C12 myoblasts is accompanied by lineage-specific changes in DNA methylation patterns. Using bisulfite sequencing and genome-wide methylated DNA- and chromatin immunoprecipitation-on-chip techniques we show that in these cells, in general, myogenic genes are enriched for RNA polymerase II and hypomethylated, whereas osteogenic genes show lower polymerase occupancy and are hypermethylated. Removal of DNA methylation marks by 5-azacytidine (5AC) treatment alters the myogenic lineage commitment of these cells and induces spontaneous osteogenic and adipogenic differentiation. This is accompanied by upregulation of key lineage-specific transcription factors. We subsequently analyzed genome-wide changes in DNA methylation and polymerase II occupancy during BMP2-induced osteogenesis. Our data indicate that BMP2 is able to induce the transcriptional program underlying osteogenesis without changing the methylation status of the genome. We conclude that DNA methylation primes C2C12 cells for myogenesis and prevents spontaneous osteogenesis, but still permits induction of the osteogenic transcriptional program upon BMP2 stimulation. Based on these results, we propose that cell type-specific DNA methylation patterns are established prior to terminal differentiation of adult progenitor cells.

Murine mesenchymal progenitor cells from different tissues differentiated via mesenchymal microspheres into the mesodermal direction

BackgroundBecause specific marker molecules for phenotypical identification of mesenchymal stem and progenitor cells are missing, the assessment of the in vitro-differentiation capacity is a prerequisite to characterize these cells. However, classical differentiation protocols are often cell-consuming and time intensive. Therefore, the establishment of novel strategies for differentiation is one topic of current efforts in stem cell biology. The goal of this study was to demonstrate the practicability of a new differentiation test using plastic adherent cell isolates from different tissues.ResultsWe introduced the mesenchymal microsphere method as a feasible time- and cell saving screening method to analyse multilineage differentiation properties of adult progenitor cells in a three-dimensional system. For this purpose we isolated, characterized and analyzed new sources of adult murine mesenchymal progenitor cells from perirenal adipose tissue and mediastinal stromal tissue in comparison to bone marrow progenitor cells. The proliferation capacity of the cells was demonstrated by determination of the daily doubling index. Although the flow cytometry analysis of undifferentiated cells revealed differences in the expression of CD marker molecules, all isolates have the capacity for multilineage differentiation following the mesenchymal microsphere protocol as well as the classical "micro mass body" protocol for chondrogenic and the monolayer cultivation protocol for osteogenic and adipogenic differentiation. Differentiation was characterized using histochemical and immunhistochemical staining as well as RT-PCR.ConclusionsWe were able to show that the mesenchymal microsphere method is an efficient test system for chondro-, osteo- and adipogenic differentiation of adult progenitor cells. The advantage of this system in comparison to classical protocols is that approximately 7 times lower cell numbers are necessary. Since classical culture procedures are time intensive because high cell numbers have to be obtained, the new differentiation method may also save cells and time in future clinical applications using human mesenchymal stromal cells.

Abstract 1818: Wnt5a Increases the Histone Demethylase Jmjd2b and Induces Epigenetic Remodelling in Adult Progenitor Cells

Wnt signalling controls the balance between stem cell proliferation and differentiation and patterning throughout development. Non-canonical Wnts, such as Wnt5a or Wnt11, enhance cardiac gene expression of endothelial progenitor cells (EPC), mesenchymal stem cells (MSC) and bone marrow mononuclear cells suggesting that non-canonical Wnts regulate cardiac commitment. However, the underlying mechanism is unclear. Using microarray analysis of Wnt5a-induced gene expression in EPC, we discovered that Wnt5a (1.0 μM, 24hours) significantly increased a variety of enzymes, which play key roles in epigenetic remodelling by modifying histone acetylation and methylation. Specifically, JMJD2B (205%), REST (311%), HDAC5 and HDAC7A (716 and 381%), YY1 (304%), and PCGF2 (352%), were significantly increased by Wnt5a. Wnt5a induced up-regulation of JMJD2B in EPC and MSC was further confirmed by real-time RT-PCR. The member of the JmjC-domain containing family, JMJD2B was recently shown to demethylate trimethylated histone H3 at lysine 9 (H3K9me3), thereby removing repressive histone modifications. Therefore, we hypothesized that increased JMJD2B might epigenetically modify gene expression, open chromatin structures at silenced promoters and modulate the differentiation of adult progenitor cells. Indeed, Wnt5a treatment reduced the repressive chromatin mark H3K9me3 as shown by Western blot and immunohistochemistry whereas global active chromatine marks (acetyl-H3 and trimethyl- H3K4) remained unchanged indicating that Wnt5a shifts the balance towards active gene expression. Furthermore, chromatin immunoprecipitation revealed that Wnt5a treatment increased the binding of JMJD2B at the promoter of the cardiac gene ANF. In line with removal of repressive chromatin marks, Wnt5a significantly increased differentiation of progenitor cells to cardiomyocytes. These data demonstrate that non-canonical Wnt5a induces epigenetic remodelling by removing repressive chromatin marks and may, thereby, sensitize adult progenitor cells for acquiring a cardiac cell fate. Further studies will investigate the specific contribution of the novel epigenetic modulator JMJD2B for adult stem cell functions.

Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells

The regulation of acetylation is central for the epigenetic control of lineage-specific gene expression and determines cell fate decisions. We provide evidence that the inhibition of histone deacetylases (HDACs) blocks the endothelial differentiation of adult progenitor cells. To define the mechanisms by which HDAC inhibition prevents endothelial differentiation, we determined the expression of homeobox transcription factors and demonstrated that HoxA9 expression is down-regulated by HDAC inhibitors. The causal involvement of HoxA9 in the endothelial differentiation of adult progenitor cells is supported by the finding that HoxA9 overexpression partially rescued the endothelial differentiation blockade induced by HDAC inhibitors. Knockdown and overexpression studies revealed that HoxA9 acts as a master switch to regulate the expression of prototypical endothelial-committed genes such as endothelial nitric oxide synthase, VEGF-R2, and VE-cadherin, and mediates the shear stress–induced maturation of endothelial cells. Consistently, HoxA9-deficient mice exhibited lower numbers of endothelial progenitor cells and showed an impaired postnatal neovascularization capacity after the induction of ischemia. Thus, HoxA9 is regulated by HDACs and is critical for postnatal neovascularization.