A Two-Stage Process for Differentiation of Wharton’s Jelly-Derived Mesenchymal Stem Cells into Neuronal-like Cells

With no permanent cure for neurodegenerative diseases, the symptoms reappear shortly after the withdrawal of medicines. A better treatment outcome can be expected if the damaged neurons are partly replaced by functional neurons and/or they are repaired using trophic factors. In this regard, safe cell therapy has been considered as a potential alternative to conventional treatment. Here, we have described a two-stage culture process to differentiate Wharton Jelly mesenchymal stem cells (WJ-MSCs) into neuronal-like cells in the presence of various cues involved in neurogenesis. The fate of cells at the end of each stage was analyzed at the morphometric, transcriptional, and translational levels. In the first stage of priming, constitutively, wingless-activated WJ-MSCs crossed the lineage boundary in favor of neuroectodermal lineage, identified by the loss of mesenchymal genes with concomitant expression of neuron-specific markers, like SOX1, PAX6, NTRK1, and NEUROD2. Neuronal-like cells formed in the second stage expressed many mature neuronal proteins like Map2, neurofilament, and Tuj1 and possessed axon hillock-like structures. In conclusion, the differentiation of a large number of neuronal-like cells from nontumorigenic and trophic factors secreting WJ-MSCs promises the development of a therapeutic strategy to treat neurodegenerative diseases

JMJD3 aids in reprogramming of bone marrow progenitor cells to hepatic phenotype through epigenetic activation of hepatic transcription factors

<div><p>The strictly regulated unidirectional differentiation program in some somatic stem/progenitor cells has been found to be modified in the ectopic site (tissue) undergoing regeneration. In these cases, the lineage barrier is crossed by either heterotypic cell fusion or direct differentiation. Though studies have shown the role of coordinated genetic and epigenetic mechanisms in cellular development and differentiation, how the lineage fate of adult bone marrow progenitor cells (BMPCs) is reprogrammed during liver regeneration and whether this lineage switch is stably maintained are not clearly understood. In the present study, we wanted to decipher genetic and epigenetic mechanisms that involve in lineage reprogramming of BMPCs into hepatocyte-like cells. Here we report dynamic transcriptional change during cellular reprogramming of BMPCs to hepatocytes and dissect the epigenetic switch mechanism of BM cell-mediated liver regeneration after acute injury. Genome-wide gene expression analysis in BM-derived hepatocytes, isolated after 1 month and 5 months of transplantation, showed induction of hepatic transcriptional program and diminishing of donor signatures over the time. The transcriptional reprogramming of BM-derived cells was found to be the result of enrichment of activating marks (H3K4me3 and H3K9Ac) and loss of repressive marks (H3K27me3 and H3K9me3) at the promoters of hepatic transcription factors (HTFs). Further analyses showed that BMPCs possess bivalent histone marks (H3K4me3 and H3K27me3) at the promoters of crucial HTFs. H3K27 methylation dynamics at the HTFs was antagonistically regulated by EZH2 and JMJD3. Preliminary evidence suggests a role of JMJD3 in removal of H3K27me3 mark from promoters of HTFs, thus activating epigenetically poised hepatic genes in BMPCs prior to partial nuclear reprogramming. The importance of JMJD3 in reprogramming of BMPCs to hepatic phenotype was confirmed by inhibiting catalytic function of the enzyme using small molecule GSK-J4. Our results propose a potential role of JMJD3 in lineage conversion of BM cells into hepatic lineage.</p></div

Binding of JMJD3 to HTFs in the presence of GSKJ4 inhibitor.

<p>Relative enrichment of H3K27me3 and JMJD3 at promoters of (A) HNF4α (B) HNF3α (C) HNF3β (D) HNF1α(E) CEBPα and (F) GATA4 determined by ChIP-qPCR. Number of experiment = 3. p value < 0.05 was considered as significant change. Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173977#pone.0173977.s017" target="_blank">S5 Table</a> for IgG control values.</p

Genome-wide transcriptional remodeling in BM-derived hepatocytes relative to Lin^- BM cells.

<p>(A) Heat map of differentially regulated hepatocyte-specific functional genes and mesenchymal and hematopoietic lineage specific genes in BM derived hepatocytes– 1 month (B1, B2) and 5 month (G1, G3) with respect to control–Lin^- BMCs (L3, L4). (B) Scatter plot analysis generated by R program comparing the log2 transformed normalized expression values of the filtered genes in Lin^- BMCs and BM-derived hepatocytes (1 month). (C) Scatter plot analysis generated by ‘R’ program comparing the log2 transformed normalized expression values of the filtered genes in primary and BM-derived hepatocytes (5 month). Number of experiment = 2 (each experiment was based on pooled cells of 3 mice).</p

Chromatin modifying enzymes in reprogramming of Lin^- BMCs to hepatocytes.

<p>(A) Expression of some chromatin modifying enzymes in BM-derived hepatocytes. Expression levels normalized to that of primary hepatocytes were used to calculate relative fold change. (B) ChIP-qPCR analyses of EZH2 at the promoters of hepatic transcription factors and hematopoietic genes in BM-derived hepatocytes. Enrichment of the marks in the immuno-precipitated samples over input samples was calculated. (C) ChIP-qPCR analyses of JMJD3 at the promoters of hepatic transcription factors and hematopoietic genes in BM-derived hepatocytes. Number of experiment = 3 (each experiment was based on pooled cells of 3 mice). Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173977#pone.0173977.s016" target="_blank">S4 Table</a> for IgG control values.</p

Generation of hepatocyte-like cells from Lin^- BM cells.

<p>(A) Immuno-histochemical analysis of recipient mice liver sections after transplantation. <i>Upper panel</i>: Images (600× magnification) showing donor derived hepatocytes (anti-GFP/donkey anti-mouse Alexa fluor 488 and anti-albumin /donkey anti-goat Alexa fluor 594). <i>Middle panel</i>: Images (630×2.8 zoom magnification) showing donor derived hepatocytes (anti-GFP/donkey anti-mouse Alexa fluor 488 and anti-HNF4α /donkey anti-goat Alexa fluor 594). <i>Lower panel</i>: IHC analysis of liver sections (630× magnification) from recipient showing donor derived hepatocytes which shows no CD45 expression (black arrow) and CD45 expressing non-parenchymal cells (white arrow). Number of experiment = 3. (B) Interphase FISH analysis of X and Y chromosome in BM-derived hepatocytes after 5 month of transplantation. The cells were analyzed for the presence of X (Red, platinum bright 550) and Y (Green, platinum bright 495) chromosomes in the nuclei (blue). (C) Cytogenetic analysis showing the percentage of donor cells that did not contain Y chromosome. Number of experiment = 3 (each experiment was based on pooled cells of 3 mice). (D) RT-qPCR analysis showing expressions of hepatic marker genes in BM-derived hepatocytes relative to expression in primary hepatocytes. Number of experiment = 3 (each experiment was based on pooled cells of 3 mice). (E) RT-qPCR analysis showing expression of various hepatic transcription factors in BM-derived hepatocytes relative to expression in primary hepatocytes. Number of experiment = 3 (each experiment was based on pooled cells of 3 mice). * No Ct.</p

Histone modifications at promoters of hepatic transcription factors in BM-derived hepatocytes.

<p>BM-derived hepatocytes were isolated after 5 months of transplantation for ChIP-qPCR analysis. Lin^-CD45⁺ and Lin^-CD45^- BMCs served as negative controls and primary hepatocytes as positive control. ChIP-qPCR analysis of (A) H3K4me3, (B) H3K9Ac, (C) H3K27me3 and (D) H3K9me3 at the promoters of hepatic transcription factors in BM-derived hepatocytes. Enrichment of the marks in the immuno-precipitated samples over input samples was calculated. Number of experiment = 3 (each experiment was based on pooled cells of 3 mice). Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173977#pone.0173977.s015" target="_blank">S3 Table</a> for IgG control values.</p

Role of antigen presenting cell invariant chain in the development of hepatic steatosis in mouse model

The role of Invariant chain (CD74 or Ii) in antigen presentation via Antigen Presenting Cells (APC), macrophage recruitment as well as survival, T cell activation and B cell differentiation has been well recognized. However, the aspect of CD74 which is involved in the development of hepatic steatosis and the pathways through which it acts remain to be studied. In this study, we investigated the role of CD74 in the inflammatory pathway and its contribution to development of hepatic steatosis. For this, wild type C57BL/6J and CD74 deficient mice (Ii<SUP>−/−</SUP> mice) were fed with high fat high fructose (HFHF) diet for 12 weeks. Chronic consumption of this feed did not develop hepatic steatosis, glucose intolerance or change in the level of immune cells in Ii<SUP>−/−</SUP> mice. Moreover, there was relatively delayed expression of genes involved in development of non alcoholic fatty liver disease (NAFLD) in HFHF fed Ii<SUP>−/−</SUP> mice as compared to that of C57BL/6J phenotype. Taken together, the data suggest that HFHF diet fed Ii<SUP>−/−</SUP> mice fail to develop hepatic steatosis, suggesting that Ii mediated pathways play a vital role in the initiation and propagation of liver inflammation