Amniotic Fluid Mesenchymal Stem Cells Research Articles

Schwann-like cells can be induction from human nestin-positive amniotic fluid mesenchymal stem cells

We examined the morphological, phenotypic, and functional characteristics of human amniotic fluid mesenchymal stem cells (AF-MSCs) differentiated towards a Schwann cell lineage. Initially, we induced human AF-MSCs into nestin-positive AF-MSCs. And then, these nestin-positive AF-MSCs were induced into floating neurospheres. After that, neurospheres were induced to differentiate into Schwann-like cells using glia growth factors. In comparison with AF-MSCs, nestin-positive AF-MSCs significantly increased the ratio of neurosphere formation and the percentage of nestin expression in the neurosphere. Differentiated AF-MSCs showed morphological changes similar to those found in Schwann cells. Expression of the Schwann cell markers was determined by immunocytochemical staining and western blotting. Furthermore, differentiated AF-MSCs could promote neurite outgrowth in co-culture with dorsal root ganglia neurons. These results suggest that conversion of human nestin-positive AF-MSCs into cells with Schwann-like cell characteristics is possible and that these cells may have the potential for future cellular therapy for peripheral neurological disorders.

Enhancement of regeneration with glia cell line–derived neurotrophic factor–transduced human amniotic fluid mesenchymal stem cells after sciatic nerve crush injury [RETRACTED

Object Human amniotic fluid–derived mesenchymal stem cells (AFMSCs) have been shown to promote peripheral nerve regeneration, and the local delivery of neurotrophic factors may additionally enhance nerve regeneration capacity. The present study evaluates whether the transplantation of glia cell line–derived neurotrophic factor (GDNF)–modified human AFMSCs may enhance regeneration of sciatic nerve after a crush injury. Methods Peripheral nerve injury was produced in Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. Either GDNF-modified human AFMSCs or human AFMSCs were embedded in Matrigel and delivered to the injured nerve. Motor function and electrophysiological studies were conducted after 1 and 4 weeks. Early or later nerve regeneration markers were used to evaluate nerve regeneration. The expression of GDNF in the transplanted human AFMSCs and GDNF-modified human AFMSCs was monitored at 7-day intervals. Results Human AFMSCs were successfully transfected with adenovirus, and a significant amount of GDNF was detected in human AFMSCs or the culture medium supernatant. Increases in the sciatic nerve function index, the compound muscle action potential ratio, conduction latency, and muscle weight were found in the groups treated with human AFMSCs or GDNF-modified human AFMSCs. Importantly, the GDNF-modified human AFMSCs induced the greatest improvement. Expression of markers of early nerve regeneration, such as increased expression of neurofilament and BrdU and reduced Schwann cell apoptosis, as well as late regeneration markers, consisting of reduced vacuole counts, increased expression of Luxol fast blue and S100 protein, paralleled the results of motor function. The expression of GDNF in GDNF-modified human AFMSCs was demonstrated up to 4 weeks; however, the expression decreased over time. Conclusions The GDNF-modified human AFMSCs appeared to promote nerve regeneration. The consecutive expression of GDNF was demonstrated in GDNF-modified human AFMSCs up to 4 weeks. These findings support a nerve regeneration scenario involving cell transplantation with additional neurotrophic factor secretion.

A microfluidic device for separation of amniotic fluid mesenchymal stem cells utilizing louver-array structures

Human mesenchymal stem cells can differentiate into multiple lineages for cell therapy and, therefore, have attracted considerable research interest recently. This study presents a new microfluidic device for bead and cell separation utilizing a combination of T-junction focusing and tilted louver-like structures. For the first time, a microfluidic device is used for continuous separation of amniotic stem cells from amniotic fluids. An experimental separation efficiency as high as 82.8% for amniotic fluid mesenchymal stem cells is achieved. Furthermore, a two-step separation process is performed to improve the separation efficiency to 97.1%. These results are based on characterization experiments that show that this microfluidic chip is capable of separating beads with diameters of 5, 10, 20, and 40 microm by adjusting the volume-flow-rate ratio between the flows in the main and side channels of the T-junction focusing structure. An optimal volume-flow-rate ratio of 0.5 can lead to high separation efficiencies of 87.8% and 85.7% for 5-microm and 10-microm beads, respectively, in a one-step separation process. The development of this microfluidic chip may be promising for future research into stem cells and for cell therapy.

Escalated regeneration in sciatic nerve crush injury by the combined therapy of human amniotic fluid mesenchymal stem cells and fermented soybean extracts, Natto

Attenuation of inflammatory cell deposits and associated cytokines prevented the apoptosis of transplanted stem cells in a sciatic nerve crush injury model. Suppression of inflammatory cytokines by fermented soybean extracts (Natto) was also beneficial to nerve regeneration. In this study, the effect of Natto on transplanted human amniotic fluid mesenchymal stem cells (AFS) was evaluated. Peripheral nerve injury was induced in SD rats by crushing a sciatic nerve using a vessel clamp. Animals were categorized into four groups: Group I: no treatment; Group II: fed with Natto (16 mg/day for 7 consecutive days); Group III: AFS embedded in fibrin glue; Group IV: Combination of group II and III therapy. Transplanted AFS and Schwann cell apoptosis, inflammatory cell deposits and associated cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. The deterioration of neurological function was attenuated by AFS, Natto, or the combined therapy. The combined therapy caused the most significantly beneficial effects. Administration of Natto suppressed the inflammatory responses and correlated with decreased AFS and Schwann cell apoptosis. The decreased AFS apoptosis was in line with neurological improvement such as expression of early regeneration marker of neurofilament and late markers of S-100 and decreased vacuole formation. Administration of either AFS, or Natto, or combined therapy augmented the nerve regeneration. In conclusion, administration of Natto may rescue the AFS and Schwann cells from apoptosis by suppressing the macrophage deposits, associated inflammatory cytokines, and fibrin deposits.

602. Development of Novel Amniotic Fluid Mesenchymal Stem Cell Therapeutic Approaches for Targeting Bladder Cancer

602. Development of Novel Amniotic Fluid Mesenchymal Stem Cell Therapeutic Approaches for Targeting Bladder Cancer

Human Amniotic Fluid Mesenchymal Stem Cells in Combination with Hyperbaric Oxygen Augment Peripheral Nerve Regeneration

Attenuation of pro-inflammatory cytokines and associated inflammatory cell deposits rescues human amniotic fluid mesenchymal stem cells (AFS) from apoptosis. Hyperbaric oxygen (HBO) suppressed stimulus-induced pro-inflammatory cytokine production in blood-derived monocyte-macrophages. Herein, we evaluate the beneficial effect of hyperbaric oxygen on transplanted AFS in a sciatic nerve injury model. Peripheral nerve injury was produced in Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. The AFS were embedded in fibrin glue and delivered to the injured site. Hyperbaric oxygen (100% oxygen, 2 ATA, 60 min/day) was administered 12 h after operation for seven consecutive days. Transplanted cell apoptosis, oxidative stress, inflammatory cell deposits and associated chemokines, pro-inflammatory cytokines, motor function, and nerve regeneration were evaluated 7 and 28 days after injury. Crush injury induced an inflammatory response, disrupted nerve integrity, and impaired nerve function in the sciatic nerve. However, crush injury-provoked inflammatory cytokines, deposits of inflammatory cytokines, and associated macrophage migration chemokines were attenuated in groups receiving hyperbaric oxygen but not in the AFS-only group. No significant increase in oxidative stress was observed after administration of HBO. In transplanted AFS, marked apoptosis was detected and this event was reduced by HBO treatment. Increased nerve myelination and improved motor function were observed in AFS-transplant, HBO-administrated, and AFS/HBO-combined treatment groups. Significantly, the AFS/HBO combined treatment showed the most beneficial effect. AFS in combination with HBO augment peripheral nerve regeneration, which may involve the suppression of apoptotic death in implanted AFS and the attenuation of an inflammatory response detrimental to peripheral nerve regeneration.

Multipotent mesenchymal stem cells from amniotic fluid originate neural precursors with functional voltage-gated sodium channels

Amniotic fluid (AF) contains stem cells with high proliferative and differentiative potential that might be an attractive source of multipotent stem cells. We investigated whether human AF contains mesenchymal stem cells (MSC) and evaluated their phenotypic characteristics and differentiation potential in vitro. AF was harvested during routine pre-natal amniocentesis at 14-16 weeks of pregnancy. AF sample pellets were plated in alpha-minimum essential medium (MEM) with 10% fetal bovine serum (FBS). We evaluated cellular growth, immunophenotype, stemness markers and differentiative potential during in vitro expansion. Neural progenitor maintenance medium (NPMM), a medium normally used for the growth and maintenance of neural stem cells, containing hFGF, hEGF and NSF-1, was used for neural induction. Twenty-seven AF samples were collected and primary cells, obtained from samples containing more than 6 mL AF, had MSC characteristics. AF MSC showed high proliferative potential, were positive for CD90, CD105, CD29, CD44, CD73 and CD166, showed Oct-4 and Nanog molecular and protein expression, and differentiated into osteoblasts, adypocytes and chondrocytes. The NPMM-cultured cells expressed neural markers and increased Na(+) channel density and channel inactivation rate, making the tetrodotoxin (TTX)-sensitive channels more kinetically similar to native neuronal voltage-gated Na(+) channels. These data suggest that AF is an important multipotent stem cell source with a high proliferative potential able to originate potential precursors of functional neurons.

Combination of G-CSF Administration and Human Amniotic Fluid Mesenchymal Stem Cell Transplantation Promotes Peripheral Nerve Regeneration

Amniotic fluid mesenchymal stem cells (AFS) harbor the potential to improve peripheral nerve injury by inherited neurotrophic factor secretion, but present the drawback of the short-term survival after transplantation. Granulocyte-colony stimulating factor (G-CSF) has a diversity of functions, including anti-inflammatory and anti-apoptotic effects. This study was conducted to evaluate whether G-CSF could augment the neuroprotective effect of transplanted AFS against peripheral nerve injury. The potential involvement of anti-inflammation/anti-apoptosis effect was also investigated. Peripheral nerve injury was produced in Sprauge-Dawley rats by crushing left sciatic nerve using a vessel clamp. The AFS were embedded in fibrin glue and delivered to the injured site. G-CSF (50 microg/kg) was administrated by intra-peritoneal injection for 7 consecutive days. Cell apoptosis, inflammatory cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. Crush injury induced inflammatory response, disrupted nerve integrity, and impaired nerve function in sciatic nerve. Crush injury-provoked inflammation was attenuated in groups receiving G-CSF but not in AFS only group. In transplanted AFS, marked apoptosis was detected and this event was reduced by G-CSF treatment. Increased nerve myelination and improved motor function were observed in AFS transplanted, G-CSF administrated, and AFS/G-CSF combined treatment groups. Significantly, the combined treatment showed the most beneficial effect. In conclusion, the concomitant treatment of AFS with G-CSF augments peripheral nerve regeneration which may involve the suppression of apoptotic death in implanted AFS and the attenuation of inflammatory response.

Post-injury regeneration in rat sciatic nerve facilitated by neurotrophic factors secreted by amniotic fluid mesenchymal stem cells

Amniotic fluid mesenchymal stem cells have the ability to secrete neurotrophic factors that are able to promote neuron survival in vitro. The purpose of this study was to evaluate the effects of neurotrophic factors secreted by rat amniotic fluid mesenchymal stem cells on regeneration of sciatic nerve after crush injury. Fifty Sprague–Dawley rats weighing 250–300 g were used. The left sciatic nerve was crushed with a vessel clamp. Rat amniotic fluid mesenchymal stem cells embedded in fibrin glue were delivered to the injured nerve. Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry were used to detect neurotrophic factors secreted by the amniotic fluid mesenchymal stem cells. Nerve regeneration was assessed by motor function, electrophysiology, histology, and immunocytochemistry studies. Positive CD29/44, and negative CD11b/45, as well as high levels of expression of brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor, ciliary neurotrophic factor (CNTF), nerve growth factor, and neurotrophin-3 (NT-3) were demonstrated in amniotic fluid mesenchymal stem cells. Motor function recovery, the compound muscle action potential, and nerve conduction latency showed significant improvement in rats treated with amniotic fluid mesenchymal stem cells. ELISA measurement in retrieved nerves displayed statistically significant elevation of CNTF and NT-3. The immunocytochemical studies demonstrated positive staining for NT-3 and CNTF in transplanted cells. The histology and immunocytochemistry studies revealed less fibrosis and a high level of expression of S-100 and glial fibrillary acid protein at the crush site. Rat amniotic fluid mesenchymal stem cells may facilitate regeneration in the sciatic nerve after crush injury. The increased nerve regeneration found in this study may be due to the neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.

Amniotic Fluid and Bone Marrow Derived Mesenchymal Stem Cells Can be Converted to Smooth Muscle Cells in the Cryo-Injured Rat Bladder and Prevent Compensatory Hypertrophy of Surviving Smooth Muscle Cells

Wound healing of the cryo-injured bladder can bring about organ remodeling because of incomplete reconstitution of depleted smooth muscle cells. Stem cell transplantation could be beneficial to improve smooth muscle cell regeneration and/or modulate the remodeling process. The repair of bladder injury using adult-type stem cells would be useful for adult urological patients but unsuited for neonatal patients, in whom major benefits are likely to derive from fetal-type stem cells. The smooth muscle cell differentiation potential of fetal-type vs adult-type stem cells was evaluated by injecting green fluorescent protein labeled mesenchymal stem cells from rat amniotic fluid or bone marrow, respectively, in cryo-injured rat bladder walls. At 30 days after transplantation only a few fetal-type or adult-type mesenchymal stem cells gave rise to enteric or vascular smooth muscle cells, whereas most mesenchymal stem cells appeared incapable of specific differentiation. In vitro co-culture experiments of smooth muscle cells with fetal-type or adult-type mesenchymal stem cells selectively labeled with distinct fluorochromes showed the presence of hybrid cells, suggesting that some mesenchymal stem cells can undergo cell fusion. Surprisingly the major effect of rat bone marrow or amniotic fluid mesenchymal stem cell transplantation seemed to be preventing cryo-injury induced hypertrophy of surviving smooth muscle cells. In this model stem cell transplantation has a limited effect on smooth muscle cell regeneration. Instead it can regulate post-injury bladder remodeling, possibly via a paracrine mechanism.

Isolation, Phenotypic, Molecular and First-Time Proteomic Characterization of Human Mesenchymal Stem Cells (MSCs) Derived from Amniotic Fluid: Comparison to Bone Marrow MSCs.

Human mesenchymal stem cells (hMSCs) constitute a population of multipotent cells, easily expanded in culture and able to give rise to many lineages. These characteristics make MSCs a very attractive tool for developing new strategies for clinical applications based on cell therapy. So far, the most common source of MSCs has been the bone marrow (BM). However, identification and characterization of alternative sources of MSCs is of great importance. One such alternative source is the amniotic fluid (AF), which can be collected during scheduled amniocentesis without any ethical concerns.To this end, in the present study, we introduced an improved protocol for isolating and clonally expanding fetal MSCs from second trimester amniotic fluid (AF) and we further characterized these cells based on theirphenotype,pluripotency,differentiation potential andproteomic profile.The AF samples were obtained during routine amniocentesis and AF-MSCs were enriched by a modified culture protocol. The isolated MSCs expanded rapidly and exhibited differentiation potential into adipocytes and osteoblasts. More importantly, we showed that these cells can differentiate in vitro not only into cell types derived from mesoderm (adipocytes and osteoblasts) and ectoderm (neural cells) but also more interestingly into endoderm (hepatocytes) derived cells. Moreover, we documented that AF-MSCs express Oct-4 transcription factor, a marker of pluripotency, and we studied for the first time its expression over different passages by real time PCR and documented that it remained constant for at least 17 doublings.An extensive characterization of the phenotypic features of AF-MSCs by using a wide range of surface markers and flow cytometry, indicated that they are positive for all the mesenchymal stem cell markers such as CD90, CD105, CD73 and CD166 and generally exhibit a similar expression pattern to the BM-MSCs.To characterize these cells in more detail, we established the first proteomic database for human AF-MSCs. Using 2D-gel electrophoresis and matrix-assisted laser desorption ionisation-time of flight-mass (MALDI-TOF) spectrometry approach, we have generated for the first time the protein map of AF MSCs, by identifying 260 proteins and directly compared this protein profile with that of MSCs derived from BM. We further performed a similar analysis for BM-MSCs, identifying 170 different proteins and generating a reference map for these cells. The comparison of the proteomic pattern from both sources was similar. In general, 140 proteins were identified in AF-MSCs related to cell growth/maintenance, metabolism/energy pathways, protein metabolism, apoptosis, signal transduction and communication as well as transcription and transport, that are not present in BM-MSCs.The approach we initiated, is expected to facilitate systematic functional studies for these multipotent cells. One such approach could be the implementation of the proteomic analysis, during differentiation of AF-MSCs to cells derived from all three germ layers as shown in our study. Data derived from these approaches are expected to clarify the therapeutic potential of the MSCs.

Enhanced regeneration in injured sciatic nerve by human amniotic mesenchymal stem cell

Objective Amniotic fluid mesenchymal stem cells (MSCs) have the potential to differentiate into neuronal stem cells in vitro. We evaluated using amniotic fluid MSCs to support or enhance the ability of the injured sciatic nerve to cross a nerve gap. Materials and methods We created a 5 mm nerve defect in Sprague Dawley rats. One group received therapy with MSCs embedded into woven oxidised regenerated cellulose gauze (Surgical; Ethicon, Somerville, NJ) and fibrin glue, while a control group received woven Surgicel and fibrin glue only. Evaluation methods included behavioural, electrophysiological and immunohistochemical studies. Results In gait analysis, the angle of the ankles in the treatment and control group were 46.4° (standard deviation [SD] = 15°) and 36° (SD = 8.2°), respectively, which was statistically significant ( p = 0.045). Five of 10 treated rats (50%) demonstrated partial foot movement, while none of the control group had any movement. The percentage amplitude of muscle compound action potential in the experimental group was 43% (SD = 12.5%) compared to 29% (SD = 8.8%) in the control group ( p = 0.038). The conduction latencies in the control and experimental groups was 2.5 ms (SD = 0.45) and 1.7 ms (SD = 0.47), respectively ( p = 0.005). Histological examination demonstrated that 70% of the treatment group achieved a maximum axon diameter percentage across the nerve gap of greater than 50%, compared with 0% in the control group. There were no differences in direction of fibre growth and fibrotic reaction between the two groups. Conclusion Amniotic fluid MSC can augment growth of injured nerve across a nerve gap. This effect may be due to neurotrophic or induction effects of the MSC interacting with Schwann cells. Further study is required to determine the underlying mechanism of this effect.