The evaluation of cartilage differentiations using transforming growth factor beta3 alone and with combination of bone morphogenetic protein-6 on adult stem cells

In our quest to standardize our formula for a clinical trial, transforming growth factor-beta3 (TGF-β3) alone and in combination with bone morphogenetic protein-6 (BMP-6) were evaluated for their effectiveness in cartilage differentiation. Bone Marrow Stem Cells (BMSCs) and Adipose Derived Stem Cells (ADSCs) were induced to chondrogenic lineage using two different media. Native chondrocytes served as positive control. ADSCs and BMSCs proved multipotency by tri-lineage differentiations. ADSC has significantly higher growth kinetics compare to Chondrocyte only p ≤ 0.05. Using TGF-β3 alone, BMSC revealed higher expressions for hyaline cartilage genes compare to ADSCs. Chondrocyte has significantly higher early chondrogenic markers expression to ADSCs and BMSCs, while BMSCs was only higher to ADSC at chondroadherin, p ≤ 0.0001. On mature chondrogenic markers, chondrocytes were significantly higher to ADSCs and BMSCs for aggrecan, collagen IX, sry (sex determining region y)-box9, collagen II and fibromodullin; and only to ADSC for collagen XI. BMSC was higher to ADSC for aggrecan and collagen IX, p ≤ 0.0001. The combination of TGF-β3 + BMP-6 revealed increased gene expressions on both BMSCs and ADSCs for early and mature chondrogenic markers, but no significance difference. For dedifferentiation markers, ADSC was significantly higher to chondrocyte for collagen I. Glycosaminoglycan evaluations with both formulas revealed that chondrocytes were significantly higher to ADSCs and BMSCs, but none was significant to each other, p ≤ 0.0001. Combination of 10 ng TGF-β3 with 10 ng of BMP-6 enhanced chondrogenic potentials of BMSCs and ADSCs compare to TGF-β3 alone. This could be the ideal cocktail for either cell’s chondrogenic induction

Safety and efficacy of human Wharton's Jelly-derived mesenchymal stem cells therapy for retinal degeneration

Purpose To investigate the safety and efficacy of subretinal injection of human Wharton’s Jelly-derived mesenchymal stem cells (hWJ-MSCs) on retinal structure and function in Royal College of Surgeons (RCS) rats. Methods RCS rats were divided into 2 groups: hWJ-MSCs treated group (n = 8) and placebo control group (n = 8). In the treatment group, hWJ-MSCs from healthy donors were injected into the subretinal space in one eye of each rat at day 21. Control group received saline injection of the same volume. Additional 3 animals were injected with nanogold-labelled stem cells for in vivo tracking of cells localisation using a micro-computed tomography (microCT). Retinal function was assessed by electroretinography (ERG) 3 days before the injection and repeated at days 15, 30 and 70 after the injection. Eyes were collected at day 70 for histology, cellular and molecular studies. Results No retinal tumor formation was detected by histology during the study period. MicroCT scans showed that hWJ-MSCs stayed localised in the eye with no systemic migration. Transmission electron microscopy showed that nanogold-labelled cells were located within the subretinal space. Histology showed preservation of the outer nuclear layer (ONL) in the treated group but not in the control group. However, there were no significant differences in the ERG responses between the groups. Confocal microscopy showed evidence of hWJ-MSCs expressing markers for photoreceptor, Müller cells and bipolar cells. Conclusions Subretinal injection of hWJ-MSCs delay the loss of the ONL in RCS rats. hWJ-MSCs appears to be safe and has potential to differentiate into retinal-like cells. The potential of this cell-based therapy for the treatment of retinal dystrophies warrants further studies

Effect of supplementation of dermal fibroblasts conditioned medium on expansion of keratinocytes through enhancing attachment

332-339In the present study in vitro expansion of human keratinocytes by supplementing dermal fibroblasts conditioned medium (DFCM) has been reported. Effect of two different DFCM acquired by culturing fibroblasts in keratinocyte-specific medium (defined keratinocytes serum free medium, DFCM-DKSFM) and fibroblast-specific serum free medium (F12: DMEM nutrient mix, DFCM-FD) have been compared. Growth kinetics of keratinocytes in terms of efficiency of cell attachment, expansion index, apparent specific growth rate and growth potential at the end of culture was evaluated in culture supplemented with DFCM-DKSFM and DFCM-FD in comparison with control i.e. DKSFM only. Results indicated that supplementation of DFCM caused significant increase in keratinocyte attachment. Efficiency of keratinocyte attachment in culture supplemented with DFCM-DKSFM was significantly higher compared to those cultured in DFCM-FD and DKSFM. In addition, the expansion index of keratinocytes in cultures supplemented with DFCM-DKSFM and DFCM-FD were 3.7 and 2.2 times higher than that of control condition even though the apparent growth rate and proliferative potential was found significantly lower. These results suggested that supplementation of DFCM enhanced expansion of keratinocyte by increasing efficiency of cell attachment, and DFCM-DKSFM provided suitable condition for in vitro expansion of keratinocytes compared to DFCM-FD and control condition

Immunohistochemistry of the injected eyes and control at day 70.

<p>Confocal microscopy at day 70 of the hWJ-MSCs and saline injected eye. Positive staining of the retinal antibody markers was detected in the hWJ-MSCs injected eyes but not in the saline injected eye. The antibodies used were Stem 121(green) for mesenchymal stem cells, Rhodopsin (red) for rod photoreceptors, MITF (green) for RPE specific markers, β tubulin (green) for immature neurons, anti-cone arrestin (red) for cone photoreceptors, PKC-α (red) for bipolar cells and recoverin (red) for cone bipolar. All slides were counterstained with DAPI (blue) to label the nucleus. Scale bar (white) represents 1000 μm.</p

Retinal histology of the injection and non-injection sites at day 70.

<p>In eye injected with hWJ-MSCs, there was a generalised preservation of outer nuclear layer (double-headed arrows) over the whole retina as shown at the site of injection (A), middle region (B) and at the site furthest away from the site of injection (C) at day 70. The site of injection was evident by a small remnant subretinal bump (red arrow). This showed that the preservation of outer nuclear layer is not limited to the injection site. Scale bar represents 50 μm.</p

Viability and cytotoxicity of hWJ-MSCs after gold nanoparticles loading.

<p>A) Representative LIVE/DEAD images of nanogold-loaded hWJ-MSCs taken at day 1, day 5 and day 10 at different concentration showed good viability where most cells were live cells which were labelled green compared to the very scant dead cells which were labelled red (indicated by white arrow). Scale bar 100μm. B) At optical density with the wavelength of 545 nm, different concentrations of 80 nm gold nanoparticles dissolved in water showed a regression (R²) of 0.99. C) The proliferation profile of hWJ-MSCs with various concentration of gold nanoparticles were evaluated over a 10-day period. Median values were represented by horizontal lines. Cell numbers on day 1, day 5 and day 10 are marked with blue circle, green square and pink triangle respectively. At day 1, there was no significant difference in cell count between control sample and samples from any of the gold nanoparticle concentrations. At day 5, the cell count was significantly reduced in the samples incubated with gold nanoparticle concentration of 1x10¹⁰ particles/ml or higher compared to the control sample (marked with asterisks). The cell count of the samples incubated with gold nanoparticles was also significantly reduced at day 10 compared to the control, but only at the concentration of 1x10¹¹ particles/ml (marked with asterisks). (p = 0.014; Mann-Whitney U test).</p

Transmission electron microscopy.

<p>Transmission electron microscopy demonstrated the uptake of gold nanoparticles (arrow heads) by the hWJ-MSCs in vitro (A). At day 5 after injection into the cell, the gold nanoparticles laden cell was located in the subretinal space (B) and some of the gold nanoparticles were seen taken up by the retinal pigment (arrow heads), (C) showed a magnified version of the gold laden cell <i>in vivo</i>.</p