Hypoxic Preconditioning Induces Neuronal Differentiation of Infrapatellar Fat Pad Stem Cells through Epigenetic Alteration.

Hypoxia is considered a key factor in cellular differentiation and proliferation, particularly during embryonic development; the process of early neurogenesis also occurs under hypoxic conditions. Apart from these developmental processes, hypoxia preconditioning or mild hypoxic sensitization develops resistance against ischemic stroke in deteriorating tissues. We therefore hypothesized that neurons resulting from hypoxia-regulated neuronal differentiation could be the best choice for treating brain ischemia, which contributes to neurodegeneration. In this study, infrapatellar fat pad (IFP), an adipose tissue present beneath the knee joint, was used as the stem cell source. IFP-derived stem cells (IFPSCs) are totally adherent and are mesenchymal stem cells. The transdifferentiation protocol involved hypoxia preconditioning, the use of hypoxic-conditioned medium, and maintenance in maturation medium with α-lipoic acid. The differentiated cells were characterized using microscopy, reverse transcription PCR, real time PCR, and immunocytochemistry. To evaluate the epigenetic reprogramming of IFPSCs to become neuron-like cells, methylation microarrays were performed. Hypoxia preconditioning stabilized and allowed for the translocation of hypoxia inducible factor 1α into the nucleus and induced achaete-scute homologue 1 and doublecortin expression. Following induction, the resultant cells expressed neuronal markers neuron-specific enolase, neurofilament-light chain, growth associated protein 43, synaptosome associated protein 25, and ß-III tubulin. The differentiated neural-lineage cells had functional gene expression pertaining to neurotransmitters, their release, and their receptors. The molecular signaling mechanisms regulated developmental neurogenesis. Furthermore, the in vitro physiological condition regulated neurotransmitter respecification or switching during IFPSC differentiation to neurons. Thus, differentiated neurons were fabricated against the ischemic region to treat neurodegenerative diseases.

Effect of passaging on the stemness of infrapatellar fat pad­derived stem cells and potential role of nucleostemin as a prognostic marker of impaired stemness.

Infrapatellar fat pad­derived stem cells (IFPSCs) are emerging as an alternative to adipose tissue­derived stem cells (ADSCs) from other sources. They are a reliable source of autologous stem cells obtained from medical waste that are suitable for use in cell­based therapy, tissue engineering and regenerative medicine. Such clinical applications require a vast number of high­quality IFPSCs. Unlike embryonic stem cells (ESCs), ADSCs and IFPSCs have limited population doubling capacity; however, in vitro expansion of primary IFPSCs through multiple passages (referred to as P) is a crucial step to acquire the desired population of cells. The present study investigated the effect of multiple passages on the stemness of IFPSCs during expansion and the possibility of predicting the loss of stemness using certain markers. IFPSCs were isolated from infrapatellar fat pad tissue resected during knee arthroplasty performed on aged patients (>65 years old). These cells from the stromal vascular fraction were serially passaged to at least to P7, and their stemness characteristics were examined at each passage. It was observed that IFPSCs maintained their spindle­shaped morphology, self­renewability and homogeneity at P2­4. Furthermore, immunostaining revealed that these cells expressed mesenchymal stem cell (CD166, CD90 and CD105) and ESC markers [Sox2, Nanog, Oct4 and nucleostemin (NS)], whereas the hematopoietic stem cell marker CD45 was absent. These cells were also able to differentiate into the three germ layer cell types, thus confirming their ability to generate clinical grade cells. The findings indicated that prolonged culture of IFPSCs (P>6) led to the loss of the stem cell proliferative marker NS, with an increased population doubling time and progression toward neuronal differentiation, acquiring a neurogenic phenotype. Additionally, IFPSCs demonstrated an inherent ability to secrete neurotrophic factors and express receptors for these factors, which is the cause of neuronal differentiation at later passages. Therefore, these findings validated NS as a prognostic indicator for impaired stemness and identified IFPSCs as a promising source for cell­based therapy, particularly for neurodegenerative diseases.

In vitro transdifferentiation of human adipose tissue-derived stem cells to neural lineage cells - a stage-specific incidence.

The present Study investigated the intrinsic ability of adipose tissue-derived stem cells (ADSCs) and their neural transdifferentiation in a stage-specific manner. Woodbury's Chemical induction was implemented with modifications to achieve neural transdifferentiation. In Group I, ADSCs were preinduced with ß-mercaptoethanol (ß-ME) and later, with neural induction medium (NIM). In Group II, ADSCs were directly treated with NIM. In Group III, a DNA methyltransferase (DNMT) inhibitor 5-azacytidine was applied to understand whether transdifferentiation is controlled by epigenetic marks. Irrespective of the presence of (ß-ME), the differentiation protocol resulted in glial-lineage cells. Group III produced poorly -differentiated neural cells with neuron-specific enolase positivity. A neuroprogenitor stage (NPC) was identified at d 11 after induction only in Group I. In other groups, this stage was not morphologically distinct. We explored the stage-specific incidence NPC, by alternatively treating them with basic fibroblast growth factor (bFGF), and antioxidants to validate if different signalling could cause varied outcomes (Group IV). They differentiated into neurons, as defined by cell polarity and expression of specific proteins. Meanwhile, neuroprogenitors exposed to NIM (Group I) produced glial-lineage cells. Further refinement and study of the occurrence and terminal differentiation of neuroprogenitors would identify a promising source for neural tissue replacement.

An innovative bioresorbable gelatin based 3D scaffold that maintains the stemness of adipose tissue derived stem cells and the plasticity of differentiated neurons.

Neural tissue engineering aims at producing a simulated environment using a matrix that is suitable to grow specialized neurons/glial cells pertaining to CNS/PNS which replace damaged or lost tissues. The primary goal of this study is to design a compatible scaffold that supports the development of neural-lineage cells which aids in neural regeneration. The fabricated, freeze-dried scaffolds consisted of biocompatible, natural and synthetic polymers: gelatin and polyvinyl pyrrolidone. Physiochemical characterization was carried out using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscopy (SEM) imaging. The 3D construct retains good swelling proficiency and holds the integrated structure that supports cell adhesion and proliferation. The composite of PVP-gelatin is blended in such a way that it matches the mechanical strength of the brain tissue. The cytocompatibility analysis shows that the scaffolds are compatible and permissible for the growth of both stem cells as well as differentiated neurons. A change in the ratios of the scaffold components resulted in varied sizes of pores giving diverse surface morphology, greatly influencing the properties of the neurons. However, there is no change in stem cell properties. Different types of neurons are characterized by the type of gene associated with the neurotransmitter secreted by them. The change in the neuron properties could be attributed to neuroplasticity. The plasticity of the neurons was analyzed using quantitative gene expression studies. It has been observed that the gelatin-rich construct supports the prolonged proliferation of stem cells and multiple neurons along with their plasticity.

The influenza A virus matrix protein 2 undergoes retrograde transport from the endoplasmic reticulum into the cytoplasm and bypasses cytoplasmic proteasomal degradation.

The matrix protein 2 (M2) is a spliced product of segment 7 genome of influenza A virus. Previous studies indicate its role in uncoating of the viral ribonucleoprotein complex during viral entry and in membrane scission while budding. Despite its crucial role in the viral life cycle, little is known about its subcellular distribution and dynamics. In this study, we have shown that the M2 protein is translocated from the membrane to the cytoplasm by a retrograde route via endosomes and the Golgi network. It utilizes retromer cargo while moving from the endosome to the trans-Golgi network and prevents endosome fusion with the lysosome. Further, M2 interacts with the endoplasmic-reticulum-resident AAA-ATPase p97 for its release into the cytoplasm. Our study also revealed that the M2 protein in the cellular milieu does not undergo ubiquitin-mediated proteasomal degradation. The migration of M2 through this pathway inside the infected cell suggests possible new roles that the M2 protein may have in the host cytoplasm, apart from its previously described functions.

Polymorphisms in the immunoregulatory genes are associated with hematopoietic recovery and increased susceptibility to bacterial infections in patients with thalassaemia major undergoing matched related hematopoietic stem cell transplantation.

In this study, the impact of polymorphisms in the genes of proinflammatory (IL-ß, TNF-α, IL-6, IFN-γ), anti-inflammatory (transforming growth factor [TGF]-ß, IL-10, IL-Ra), and other immunoregulatory factors (FcγRIIa, NOS3) along with the conventional risk factors on the rate of hematopoietic recovery and first episodes of bacterial, viral, or invasive fungal infections in 102 patients with ß-thalassaemia major who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) with relatively uniform protocols at our center from June 1995 to June 2004 with a minimum follow-up of at least 2 years were studied retrospectively for 180 days after hematopoietic stem cell transplantation (HSCT). Our data show that (1) donor IL-1RN∗2/2 (hazard ratio [HR], 2.4; 95% confidence interval [CI], 1.17-5.09; P = .018) and FCγRIIA +4481G/G genotypes (HR, 3.1; 95% CI, 1.56-6.31; P = .001) increased the incidence of bacterial infection; (2) fungal infection was increased in recipients with whose donors had IFN-γ +874T/T genotype (HR, 3.8; 95% CI, 1.08-13.62; P = .037); (3) time to neutrophil recovery was shorter in splenectomized patients (HR, 3.1; 95% CI, 1.70-5.64; P < .001), donors without IL-10 -1082A, -819T, and -592A haplotype (HR, 1.6; 95% CI, 1.02-2.39; P = .039), and recipients with IFN-γ +874A/A genotype (HR, 1.6; 95% CI, 1.05-2.56; P = .029); and (4) time to platelet recovery was shorter in patients with IL-10 -1082A/A genotype (HR, 1.8; 95% CI, 1.14-2.68; P = .010) and with donors having TNF-α -308G/G genotypes (HR, 1.8; 95% CI, 1.06-2.93; P = .028). These data suggest that outcome after allogeneic stem cell transplantation could be affected by many factors. The mechanisms by which they bring about such impact needs further evaluation.

Plasmacytoid dendritic cell count on day 28 in HLA-matched related allogeneic peripheral blood stem cell transplant predicts the incidence of acute and chronic GVHD.

Dendritic cells (DC) are antigen-presenting cells involved in induction and regulation of immune responses. We investigated the impact of the number of infused and day 28 dendritic cells on the development of acute and chronic GVHD (aGVHD, cGVHD). Monocytoid (MC) and plasmacytoid (PC) dendritic cells were characterized as lin(-)HLA-DR(+)CD11c(+) and lin(-)HLA-DR(+)CD123(+), respectively. Sixty-eight consecutive patients who underwent HLA matched related granuloyte-colony stimulating factor (G-CSF) mobilized allogeneic PBSCT, from February 2005 to May 2006, were included in the analysis. Twenty-three patients developed aGVHD (grade II-IV) and 21 patients had cGVHD. On a univariate analysis the day 28 total DC and the day 28 MC and PC dendritic cells as continuous variables were significantly associated with development of aGVHD and cGVHD. Using an ROC plot analysis a cutoff value for total DC = 10.7/microL, MC = 9.7/microL, and PC = 4.5/microL on day 28 gave the highest likelihood ratios for aGVHD (2.7, 2.14, and 3.29, respectively). On a multivariate analysis, a low day 28 PC (