Abstract The recent progress in stem cell techniques has broadened the horizon for in vitro disease modeling. For desired in vivo like phenotypes, not only correct cell type specification will be ...critical, the microenvironmental context will be essential to achieve relevant responses. We demonstrate how a three dimensional (3D) culture of stem cell derived neurons can induce in vivo like responses related to Alzheimer's disease, not recapitulated with conventional 2D cultures. To acquire a neural population of cells we differentiated neurons from neuroepithelial stem cells, derived from induced pluripotent stem cells. p21-activated kinase mediated sensing of Aβ oligomers was only possible in the 3D environment. Further, the 3D phenotype showed clear effects on F-actin associated proteins, connected to the disease processes. We propose that the 3D in vitro model has higher resemblance to the AD pathology than conventional 2D cultures and could be used in further studies of the disease.
In order to investigate the effects of bone marrow‐derived MSCs (mesenchymal stem cells) in reversing liver fibrosis and to determine their possible mechanism of action, mouse MSCs were infused into ...the tail vein of a CCl4 injection mouse chronic model. MSCs caused a decrease in liver fibrosis histopathologically, 4 weeks after transplantation. The reduction in liver collagen was confirmed by quantitative analysis. Moreover, lipid peroxidation in the CCl4/MSC group decreased significantly. Quantitative RT (reverse transcription)‐PCR analysis showed administration of MSCs has a significant antifibrotic effect as evidenced by the decrease in expression of liver collagen and increase in MMP13 (matrix metalloproteinase 13) in the CCl4/MSC group when compared with the CCl4 group, 4 weeks after transplantation. The expression of αSMA (smooth muscle actin) and TIMP1 was also down‐regulated in the CCl4/MSC group. Additionally, the expression of MMP9 was significantly up‐regulated in the CCl4‐treated group; however, there was no significant change after MSC injection. Few engrafted cells in the recipient liver and were able to differentiate into albumin‐positive cells. In conclusion, MSCs can enhance recovery of a CCl4‐injured mouse liver through their influence in reducing collagen deposition by possibly affecting expression of MMPs.
Induced pluripotent stem cells (iPSCs) from patients are an attractive disease model to study tissues with poor accessibility such as the brain. Using this approach, we and others have shown that ...trisomy 21 results in genome-wide transcriptional dysregulations. The effects of loss of genes on chromosome 21 is much less characterized. Here, we use patient-derived neural cells from an individual with neurodevelopmental delay and a ring chromosome 21 with two deletions spanning 3.8 Mb at the terminal end of 21q22.3, containing 60 protein-coding genes. To investigate the molecular perturbations of the partial monosomy on neural cells, we established patient-derived iPSCs from fibroblasts retaining the ring chromosome 21, and we then induced iPSCs into neuroepithelial stem cells. RNA-Seq analysis of NESCs with the ring chromosome revealed downregulation of 18 genes within the deleted region together with global transcriptomic dysregulations when compared to euploid NESCs. Since the deletions on chromosome 21 represent a genetic "contrary" to trisomy of the corresponding region, we further compared the dysregulated transcriptomic profile in with that of two NESC lines with trisomy 21. The analysis revealed opposed expression changes for 23 genes on chromosome 21 as well as 149 non-chromosome 21 genes. Taken together, our results bring insights into the effects on the global and chromosome 21 specific gene expression from a partial monosomy of chromosome 21qter during early neuronal differentiation.
The etiology of hereditary ataxia syndromes is heterogeneous, and the mechanisms underlying these disorders are often unknown. Here, we utilized exome sequencing in two siblings with progressive ...ataxia and muscular weakness and identified a novel homozygous splice mutation (c.3020-1G > A) in neurofascin (
NFASC
). In RNA extracted from fibroblasts, we showed that the mutation resulted in inframe skipping of exon 26, with a deprived expression of the full-length transcript that corresponds to
NFASC
isoform NF186. To further investigate the disease mechanisms, we reprogrammed fibroblasts from one affected sibling to induced pluripotent stem cells, directed them to neuroepithelial stem cells and finally differentiated to neurons. In early neurogenesis, differentiating cells with selective depletion of the NF186 isoform showed significantly reduced neurite outgrowth as well as fewer emerging neurites. Furthermore, whole-cell patch-clamp recordings of patient-derived neuronal cells revealed a lower threshold for openings, indicating altered Na
+
channel kinetics, suggesting a lower threshold for openings as compared to neuronal cells without the
NFASC
mutation. Taken together, our results suggest that loss of the full-length
NFASC
isoform NF186 causes perturbed neurogenesis and impaired neuronal biophysical properties resulting in a novel early-onset autosomal recessive ataxia syndrome.
There are limited data available on the effect of a physicochemical microenvironment on mesenchymal stem cell (MSC) differentiation and repopulation of the liver. Therefore, in this study nanofibers ...have been used to better differentiate and maintain the function and engraftment of differentiating MSCs both in vitro and in vivo. Mouse MSCs were differentiated into early (day 18) and late (day 36) hepatocyte-like cells (HLCs) in the presence or absence of ultraweb nanofibers (nano
+
and nano
−
) and their transplantation for recovery in mice with CCl
4
induced hepatic fibrosis was investigated. In the nano
+
group, hepatocyte markers-ALB and HNF4α- were elevated in a time-dependent manner; however, those were similar levels or slightly decreased in the nano
−
group from day 18 to 36. Ultrastructural studies of the differentiated cells revealed some similarities to hepatocytes. Urea production, secretion of albumin and α-fetoprotein, and metabolic activity of the CYP450 enzymes were significantly increased within in vitro differentiated HLCs on nanofibers at day 36. MSCs, early and late HLCs in both nano
−
and nano
+
culture conditions that were transplanted by an intravenous route caused a decrease in liver fibrosis when engrafted in the recipient liver and were able to differentiate into functional hepatocytes (ALB
+
), except for late HLCs in the nano
−
group. Late HLCs transplanted in the nano
+
group were more effective in rescuing liver failure, enhancing serum ALB, homing transplanted cells and undergoing functional engraftment than the other groups. These results showed that topographic properties of nanofibers enhance differentiation of HLCs from MSCs and maintain their function in long-term culture, which has implications for cell therapies.
Down syndrome (DS) or trisomy 21 (T21) is a leading genetic cause of intellectual disability. To gain insights into dynamics of molecular perturbations during neurogenesis in DS, we established a ...model using induced pluripotent stem cells (iPSC) with transcriptome profiles comparable to that of normal fetal brain development. When applied on iPSCs with T21, transcriptome and proteome signatures at two stages of differentiation revealed strong temporal dynamics of dysregulated genes, proteins and pathways belonging to 11 major functional clusters. DNA replication, synaptic maturation and neuroactive clusters were disturbed at the early differentiation time point accompanied by a skewed transition from the neural progenitor cell stage and reduced cellular growth. With differentiation, growth factor and extracellular matrix, oxidative phosphorylation and glycolysis emerged as major perturbed clusters. Furthermore, we identified a marked dysregulation of a set of genes encoded by chromosome 21 including an early upregulation of the hub gene
APP
, supporting its role for disturbed neurogenesis, and the transcription factors
OLIG1
,
OLIG2
and
RUNX1
, consistent with deficient myelination and neuronal differentiation. Taken together, our findings highlight novel sequential and differentiation-dependent dynamics of disturbed functions, pathways and elements in T21 neurogenesis, providing further insights into developmental abnormalities of the DS brain.
Endogenous retroviruses (ERVs), which make up 8% of the human genome, have been proposed to participate in the control of gene regulatory networks. In this study, we find a region- and developmental ...stage-specific expression pattern of ERVs in the developing human brain, which is linked to a transcriptional network based on ERVs. We demonstrate that almost 10,000, primarily primate-specific, ERVs act as docking platforms for the co-repressor protein TRIM28 in human neural progenitor cells, which results in the establishment of local heterochromatin. Thereby, TRIM28 represses ERVs and consequently regulates the expression of neighboring genes. These results uncover a gene regulatory network based on ERVs that participates in control of gene expression of protein-coding transcripts important for brain development.
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•Stage- and region-specific expression of ERVs during human brain development•TRIM28 binds to ERVs and induces hetereochromatin in human neural progenitor cells•Knockdown of TRIM28 in hNPCs results in the upregulation of ERV expression•Protein-coding genes located near upregulated ERVs are upregulated
Brattås et al. report that endogenous retroviruses are bound by TRIM28 in human neural progenitor cells. This results in the establishment of local heterochromatin that affects nearby gene expression, suggesting a role for endogenous retroviruses in the control of transcriptional networks in the developing human brain.
Embryonic stem cells (ESCs) are considered one promising new approach to generate a transplantable cell source for the treatment of liver diseases. Because traditional methods, such as the initial ...formation of embryoid body in the presence of serum result in all three germ layer derivatives, strategies have been utilized that favor cell-specific differentiation to generate more uniformity. Here, we have presented the use of a multistep protocol with growth factors in a serum-free adherent culture configuration to mediate the hepatocyte differentiation of human ESCs (hESCs). The differentiated cells exhibited characteristic hepatocyte morphology, ultrastructure, and expressed hepatic-related genes as shown by reverse transcription-polymerase chain reaction and displayed antibody detectable expression of markers specific for hepatic maturation. These hepatocyte-like cells also demonstrated evidence of albumin and α-fetoprotein secretion, glycogen storage, urea production, uptake of low-density lipoprotein, and indocyanine green. Therefore, we propose that the hepatocyte-like cells derived from hESCs by the present method may provide a useful model for the studies of key events during early liver development and a potential source of drug screening and transplantable cells for cell-replacement therapies.
Abstract Human pluripotent stem cells (hPSCs) can undergo unlimited self-renewal and have the capacity to differentiate into all somatic cell types, and are therefore an ideal source for the ...generation of cells and tissues for research and therapy. To realize this potential, defined cell culture systems that allow expansion of hPSCs and subsequent controlled differentiation, ideally in an implantable three-dimensional (3D) matrix, are required. Here we mimic spider silk – Nature's high performance material – for the design of chemically defined 2D and 3D matrices for cell culture. The silk matrices do not only allow xeno-free long-term expansion of hPSCs but also differentiation in both 2D and 3D. These results show that biomimetic spider silk matrices enable hPSC culture in a manner that can be applied for experimental and clinical purposes.
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