In vitro differentiation of adipose-tissue-derived mesenchymal stem cells into neural retinal cells through expression of human PAX6 (5a) gene

Abstract

The neural retina is subjected to various degenerative conditions. Regenerative stem-cell-based therapy holds great promise for treating severe retinal degeneration diseases, although many drawbacks remain to be overcome. One important problem is to gain authentically differentiated cells for replacement. Paired box 6 protein (5a) (PAX6 (5a)) is a highly conserved master control gene that has an essential role in the development of the vertebrate visual system. Human adipose-tissue-derived stem cell (hADSC) isolation was performed by using fat tissues and was confirmed by the differentiation potential of the cells into adipocytes and osteocytes and by their surface marker profile. The coding region of the human PAX6 (5a) gene isoform was cloned and lentiviral particles were propagated in HEK293T. The differentiation of hADSCs into retinal cells was characterized by morphological characteristics, quantitative real-time reverse transcription plus the polymerase chain reaction (qPCR) and immunocytochemistry (ICC) for some retinal cell-specific and retinal pigmented epithelial (RPE) cell-specific markers. hADSCs were successfully isolated. Flow cytometric analysis of surface markers indicated the high purity (~97 %) of isolated hADSCs. After 30 h of post-transduction, cells gradually showed the characteristic morphology of neuronal cells and small axon-like processes emerged. qPCR and ICC confirmed the differentiation of some neural retinal cells and RPE cells. Thus, PAX6 (5a) transcription factor expression, together with medium supplemented with fibronectin, is able to induce the differentiation of hADSCs into retinal progenitors, RPE cells and photoreceptors.

Good manufacturing practice-compliant isolation and culture of human adipose derived stem cells

“…These expanded ADSCs exhibit the MSC phenotype (Bourin et al, 2013;Zimmerlin et al, 2013), as is evidenced by the expression of MSC markers such as CD44, CD73, CD90 and CD105. These cells also successfully differentiate into adipocytes, osteoblasts, and chondrocytes, and could be trans-differentiated into other mesoderm cell types such as insulin producing cells (Karaoz et al, 2013;Moshtagh et al, 2013), hepatocytes (Aurich et al, 2009;Lee et al, 2012;Okura et al, 2010), and neuronal-like cells (Cardozo et al, 2010;Rezanejad et al, 2014). (Sandor et al, 2014), non-revascularizable critical limb ischemia (Bura et al, 2014), acute myocardial infarction and heart failure (Panfilov et al, 2013), complex perianal fistula in Crohn's disease (de la Portilla et al, 2013;Garcia-Olmo et al, 2009), and chronic myocardial ischemia (Qayyum et al, 2012).…”

Progress of stem/progenitor cell-based therapy for retinal degeneration

“…(2) Non-ocular-derived stem cells (with the potential to self-renew and produce different cells including RPE, photoreceptors, etc. ), include embryonic stem cells (ESCs) [9], induced pluripotent stem cells (iPSCs) [10], mesenchymal stromal cells (MSCs) (particularly bone marrow mesenchymal stromal cells (BM-MSCs) [11] and adipose-derived stromal cells (ADSCs) [12]).…”

“…It was reported that human ADSCs have the capability to differentiate into neural retinal cells in vitro with paired box 6 protein (5a) gene expression [12]. In the laboratory of Li et al, subretinal transplantation of GFP-labelled human ADSCs into RCS rats effectively enhanced the survival rate of retinal cells, delayed RD and gave rise to increased visual function through the secretion of vascular endothelial growth factor, [110].…”

Storage effect on viability and biofunctionality of human adipose tissue-derived stromal cells

“…These cells bear therapeutic potential in regenerative medicine and tissue engineering applications [3e5]. Currently, ASCs are widely used in treatment of a variety of tissue injuries [6,7] and degenerative diseases [8,9] and have been assessed in a number of clinical trials [5,10,11]. ASCs can be isolated from various sources of adipose tissues, which are distributed across multiple locations in the body.…”