Stem Cell Differentiation

Stem/progenitor cell-based therapeutic approach in clinical practice has been an elusive dream in medical sciences, and improvement of stem cell homing is one of major challenges in cell therapy programs. Stem/progenitor cells have a homing response to injured tissues/organs, mediated by interactions of chemokine receptors expressed on the cells and chemokines secreted by the injured tissue. For improvement of directed homing of the cells, many techniques have been developed either to engineer stem/progenitor cells with higher amount of chemokine receptors (stem cell-based strategies) or to modulate the target tissues to release higher level of the corresponding chemokines (target tissue-based strategies). This review discusses both of these strategies involved in the improvement of stem cell homing focusing on mesenchymal stem cells as most frequent studied model in cellular therapies.

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 adiposetissue-
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.

Cutaneous wound healing is a complex type of biological event involving proliferation, differentiation, reprograming, trans/de-differentiation , recruitment, migration, and apoptosis of a number of cells (keratinocytes, fibroblasts, endothelial cells, nerve cells and stem cells) to regenerate a multi-layered tissue that is damaged by either internal or external factors. The exact regeneration mechanism of damaged skin is still unknown but the epithelial and other kinds of stem cells located in skin play crucial roles in the healing process. In this work, a co-culture model composed of adipose derived mesenchymal stem cells and keratinocytes was developed to understand the cellular differentiation behaviour in wound healing. Human mesenchymal stem cells were isolated from waste lipoaspirates. Keratinocytes were isolated from neonatal rats skin as well from human adult skin. Both types of cells were cultured and their culturing behaviour was observed microscopically under regular intervals of time. The identity of both cells was confirmed by flow cytometry and qRT-PCR. Cells were co-cultured under the proposed co-culturing model and the model was observed for 7, 14 and 21 days. The cellular behaviour was studied based on change in morphology, colonization, stratification, migration and expression of molecular markers. Expression of molecular markers was studied at transcriptional level and change in cellular morphology and migration capabilities was observed under the invert microscope regularly. Successfully isolated and characterized mesenchymal stem cells were found to express keratinocyte lineage markers i.e. K5, K10, K14, K18, K19 and Involucrin when co-cultured with keratinocytes after 14 and 21 days. Their expression was found to increase by increasing the time span of cell culturing. The keratinocyte colonies started to disappear after 10 days of culturing which might be due to stratification process initiated by possibly transdifferentiated stem cells. It can be concluded that mesenchymal stem cells can regenerate the damaged skin if transplanted to damaged area but for their successful differentiation and enhanced regeneration, they need a population of keratinocytes in situ which need further experiments for validation of co-culture model and its potential for being used in clinics.

Gene amplifications are mostly an attribute of tumor cells and drug resistant
cells. Recently, we provided evidence for gene amplifications during differentiation
of human and mouse neural progenitor cells. Here, we report gene amplifications in
differentiating mouse myoblasts (C2C12 cells) covering a period of 7 days including
pre-fusion, fusion and post-fusion stages. After differentiation induction we found an
increase in copy numbers of CDK4 gene at day 3, of NUP133 at days 4 and 7, and of
MYO18B at day 4. The amplification process was accompanied by gamma-H2AX foci
that are indicative of double stand breaks. Amplifications during the differentiating
process were also found in primary human myoblasts with the gene CDK4 and NUP133
amplified both in human and mouse myoblasts. Amplifications of NUP133 and CDK4
were also identified in vivo on mouse transversal cryosections at stage E11.5. In the
course of myoblast differentiation, we found amplifications in cytoplasm indicative of
removal of amplified sequences from the nucleus. The data provide further evidence
that amplification is a fundamental mechanism contributing to the differentiation
process in mammalians.