Human Hair Outer Root Sheath Cells and Platelet-Lysis Exosomes Promote Hair Inductivity of Dermal Papilla Cell

BACKGROUND@#Hair loss is a prevalent medical problem in both men and women. Maintaining the potential hair inductivityof dermal papilla cells (DPCs) during cell culture is the main factor in hair follicle morphogenesis and regeneration. Thepresent study was conducted to compare the effects of different concentrations of human hair outer root sheath cell(HHORSC)and platelet lysis (PL) exosomes to maintain hair inductivity of the human dermal papilla cells (hDPCs). @*METHODS@#In this study, hDPCs and HHORSCswere isolated from healthy hair samples. Specific markers of hDPCs (versican,a-SMA) and HHORSCs (K15) were evaluated using flow cytometric and immunocytochemical techniques. The exosomes wereisolated fromHHORSCsand PL with ultracentrifugation technique.Western blot was used to detect specific markers of HHORSCsand PL exosomes. Particle size and distribution of the exosomes were analyzed by NanoSight dynamic light NanoSight DynamicLight Scattering. Different methods such as proliferation test (MTS assay), migration test (Transwell assay) were used to evaluatethe effects of different concentrations of exosomes (2,550,100 lg/ml) derived from HHORSC and PL on hDPCs. Expression ofspecific genes in the hair follicle inductivity, including ALP, versican and a-SMA were also evaluated using real time-PCR. @*RESULTS@#The flow cytometry of the specific cytoplasmic markers of the hDPCs and HHORSCs showed expression ofversican (77%), a-SMA (55.2%) and K15 (73.2%). The result of particle size and distribution of the exosomes wereanalyzed by NanoSight dynamic light NanoSight Dynamic Light Scattering, which revealed the majority of HHORSC andPL exosomes were 30–150 nm. For 100 lg/ml of HHORSC exosomes, the expressions of ALP, versican and a-SMAproteins respectively increased by a factor of 2.1, 1.7and 1.3 compared to those in the control group. @*CONCLUSION@#In summary, we applied HHORSC exosomes as a new method to support hair inductivity of dermalpapilla cells and improve the outcome for the treatment of hair loss.

Human Hair Outer Root Sheath Cells and Platelet-Lysis Exosomes Promote Hair Inductivity of Dermal Papilla Cell

BACKGROUND@#Hair loss is a prevalent medical problem in both men and women. Maintaining the potential hair inductivityof dermal papilla cells (DPCs) during cell culture is the main factor in hair follicle morphogenesis and regeneration. Thepresent study was conducted to compare the effects of different concentrations of human hair outer root sheath cell(HHORSC)and platelet lysis (PL) exosomes to maintain hair inductivity of the human dermal papilla cells (hDPCs). @*METHODS@#In this study, hDPCs and HHORSCswere isolated from healthy hair samples. Specific markers of hDPCs (versican,a-SMA) and HHORSCs (K15) were evaluated using flow cytometric and immunocytochemical techniques. The exosomes wereisolated fromHHORSCsand PL with ultracentrifugation technique.Western blot was used to detect specific markers of HHORSCsand PL exosomes. Particle size and distribution of the exosomes were analyzed by NanoSight dynamic light NanoSight DynamicLight Scattering. Different methods such as proliferation test (MTS assay), migration test (Transwell assay) were used to evaluatethe effects of different concentrations of exosomes (2,550,100 lg/ml) derived from HHORSC and PL on hDPCs. Expression ofspecific genes in the hair follicle inductivity, including ALP, versican and a-SMA were also evaluated using real time-PCR. @*RESULTS@#The flow cytometry of the specific cytoplasmic markers of the hDPCs and HHORSCs showed expression ofversican (77%), a-SMA (55.2%) and K15 (73.2%). The result of particle size and distribution of the exosomes wereanalyzed by NanoSight dynamic light NanoSight Dynamic Light Scattering, which revealed the majority of HHORSC andPL exosomes were 30–150 nm. For 100 lg/ml of HHORSC exosomes, the expressions of ALP, versican and a-SMAproteins respectively increased by a factor of 2.1, 1.7and 1.3 compared to those in the control group. @*CONCLUSION@#In summary, we applied HHORSC exosomes as a new method to support hair inductivity of dermalpapilla cells and improve the outcome for the treatment of hair loss.

Hair Follicle as a Source of Pigment-Producing Cells for Treatment of Vitiligo: An Alternative to Epidermis?

To discuss the advantages and limitations of hair follicle-derived cell transplantation (FCT) in vitiligo, compared to the epidermal cell transplantation (ECT), and the knowledge gap which is required to be bridged. The papers relevant to the purpose was reviewed. Surgical approaches for treating vitiligo are based on the idea of replenishing lost melanocytes.Skin and hair follicles as the main sources of melanocytes have been applied for this purpose transferring the whole tissue or tissue-derived cell suspension to the vitiligo lesions. Considering the differences between hair follicle and epidermis in terms of the constituting cell populations, phenotype and function of melanocytes, and micro-environmental factors, different response of vitiligo patients to treatment with FCT or ECT would be expected theoretically. However, there is currently a lack of evidence on such a difference. However, ECT appears to be a more feasible, less time-consuming, and more comfortable treatment for both physicians and patients. Although the current evidence has not shown a significant difference between ECT and FCT in terms of efficacy, ECT appears to be more feasible specifically in the treatment of large lesions. However, further randomized controlled clinical trials with larger sample sizes and longer follow-up durations are required to be conducted to draw a definite conclusion on comparing FCT with ECT in terms of the safety, efficacy, durability of the therapeutic effects, and indications in vitiligo patients.

Hair Follicle as a Source of Pigment-Producing Cells for Treatment of Vitiligo: An Alternative to Epidermis?

To discuss the advantages and limitations of hair follicle-derived cell transplantation (FCT) in vitiligo, compared to the epidermal cell transplantation (ECT), and the knowledge gap which is required to be bridged. The papers relevant to the purpose was reviewed. Surgical approaches for treating vitiligo are based on the idea of replenishing lost melanocytes.Skin and hair follicles as the main sources of melanocytes have been applied for this purpose transferring the whole tissue or tissue-derived cell suspension to the vitiligo lesions. Considering the differences between hair follicle and epidermis in terms of the constituting cell populations, phenotype and function of melanocytes, and micro-environmental factors, different response of vitiligo patients to treatment with FCT or ECT would be expected theoretically. However, there is currently a lack of evidence on such a difference. However, ECT appears to be a more feasible, less time-consuming, and more comfortable treatment for both physicians and patients. Although the current evidence has not shown a significant difference between ECT and FCT in terms of efficacy, ECT appears to be more feasible specifically in the treatment of large lesions. However, further randomized controlled clinical trials with larger sample sizes and longer follow-up durations are required to be conducted to draw a definite conclusion on comparing FCT with ECT in terms of the safety, efficacy, durability of the therapeutic effects, and indications in vitiligo patients.

Systemic infusion of autologous adipose tissue-derived mesenchymal stem cells in peritoneal dialysis patients: feasibility and safety

Objective: Using mesenchymal stem cells [MSCs] is regarded as a new therapeutic approach for improving fibrotic diseases.The aim of this study to evaluate the feasibility and safety of systemic infusion of autologous adipose tissue-derived MSCs [AD-MSCs] in peritoneal dialysis [PD] patients with expected peritoneal fibrosis

Materials and Methods: This study was a prospective, open-label, non-randomized, placebo-free, phase I clinical trial. Case group consisted of nine eligible renal failure patients with more than two years of history of being on PD. Autologous AD-MSCs were obtained through lipoaspiration and expanded under good manufacturing practice conditions. Patients received 1.2 +/- 0.1×106 cell/kg of AD-MSCs via cubital vein and then were followed for six months at time points of baseline, and then 3 weeks, 6 weeks, 12 weeks, 16 weeks and 24 weeks after infusion. Clinical, biochemical and peritoneal equilibration test [PET] were performed to assess the safety and probable change in peritoneal solute transport parameters

Results: No serious adverse events and no catheter-related complications were found in the participants. 14 minor reported adverse events were self-limited or subsided after supportive treatment. One patient developed an episode of peritonitis and another patient experienced exit site infection, which did not appear to be related to the procedure. A significant decrease in the rate of solute transport across peritoneal membrane was detected by PET [D/P cr=0.77 vs. 0.73, P=0.02]

Conclusion: This study, for the first time, showed the feasibility and safety of AD-MSCs in PD patients and the potentials for positive changes in solute transport. Further studies with larger samples, longer follow-up, and randomized blind control groups to elucidate the most effective route, frequency and dose of MSCs administration, are necessary [Registration Number: IRCT2015052415841N2]

Establishment of a protocol for in vitro culture of cardiogenic mesodermal cells derived from human embryonic stem cells

Objective: Cardiovascular progenitor cells [CPCs] are introduced as one of the promising cell sources for preclinical studies and regenerative medicine. One of the earliest type of CPCs is cardiogenic mesoderm cells [CMCs], which have the capability to generate all types of cardiac lineage derivatives. In order to benefit from CMCs, development of an efficient culture strategy is required. We aim to explore an optimized culture condition that uses human embryonic stem cell [hESC]-derived CMCs

Materials and Methods: In this experimental study, hESCs were expanded and induced toward cardiac lineage in a suspension culture. Mesoderm posterior 1-positive [MESP1+] CMCs were subjected to four different culture conditions: i. Suspension culture of CMC spheroids, ii. Adherent culture of CMC spheroids, iii. Adherent culture of single CMCs using gelatin, and iv. Adherent culture of single CMCs using Matrigel

Results: Although, we observed no substantial changes in the percentage of MESP1+ cells in different culture conditions, there were significantly higher viability and total cell numbers in CMCs cultured on Matrigel [condition iv] compared to the other groups. CMCs cultivated on Matrigel maintained their progenitor cell signature, which included the tendency for cardiogenic differentiation

Conclusion: These results showed the efficacy of an adherent culture on Matrigel for hESC-derived CMCs, which would facilitate their use for future applications

Safety, feasibility of intravenous and intrathecal injection of autologous bone marrow derived mesenchymal stromal cells in patients with amyotrophic lateral sclerosis: An open label phase I clinical trial

Objective: Amyotrophic lateral sclerosis [ALS] is the most severe disorder within the spectrum of motor neuron diseases [MND] that has no effective treatment and a progressively fatal outcome. We have conducted two clinical trials to assess the safety and feasibility of intravenous [IV] and intrathecal [IT] injections of bone marrow derived mesenchymal stromal cells [BM-MSCs] in patients with ALS

Materials and Methods: This is an interventional/experimental study. We enrolled 14 patients that met the following inclusion criteria: definitive diagnosis of sporadic ALS, ALS Functional Rating Scale [ALS-FRS] .24, and .40% predicted forced vital capacity [FVC]. All patients underwent bone marrow [BM] aspiration to obtain an adequate sample for cell isolation and culture. Patients in group 1 [n=6] received an IV and patients in group 2 [n=8] received an IT injection of the cell suspension. All patients in both groups were followed at 24 hours and 2, 4, 6, and 12 months after the injection with ALS-FRS, FVC, laboratory tests, check list of side effects and brain/spinal cord magnetic resonance imaging [MRI]. In each group, one patient was lost to follow up one month after cell injection and one patient from IV group died due to severe respiratory insufficiency and infection

Results: During the follow up there were no reports of adverse events in terms of clinical and laboratory assessments. In MRI, there was not any new abnormal finding. The ALS-FRS score and FVC percentage significantly reduced in all patients from both groups

Conclusion: This study has shown that IV and IT transplantation of BM-derived stromal cells is safe and feasible [Registration numbers: NCT01759797 and NCT01771640]

Isolation, characterization and osteogenic potential of mouse digit tip blastema cells in comparison with bone marrow-derived mesenchymal stem cells in vitro

Objective: limb regeneration mediated by blastema cells [BlCs] in mammals is limited to the digit tips of neonates. Due to the lack of access to BlCs in adults and the difficulty in isolating and expanding BlCs from neonates, the use of a cellular population with similar features of BlCs would be a valuable strategy to direct a non-regenerative wound towards regeneration. In this study, we have initially isolated and cultured BlCs, and explored their characteristics in vitro. Next, we compared the capability of bone marrow-derived mesenchymal stem cells [BM-MSCs] as an alternative accessible cell source to BlCs for regeneration of appendages

Materials and Methods: in this experimental study, BM-MSCs were isolated from BM and we obtained BlCs from the neonatal regenerating digit tip of C57B/6 mice. The cells were characterized for expressions of cell surface markers by flow cytometry. Quantitative-reverse transcription polymerase chain reaction [qRT-PCR] and lineage-specific staining were used to assess their ability to differentiate into skeletal cell lineages. The colony forming ability, proliferation, alkaline phosphatase [ALP] activity, calcium content, and osteogenic gene expression were evaluated in both BMMSCs and BlCs cultures at days 7, 14, and 21

Results: qRT-PCR analysis revealed that the cells from both sources readily differentiated into mesodermal lineages. There was significantly higher colony forming ability in BM-MSCs compared to BlCs [P<0.05]. Alizarin red staining [ARS], calcium, and the ALP assay showed the same degree of mineral deposition in both BlCs and BM-MSCs. Gene expression levels of osteblastic markers indicated similar bone differentiation capacity for both BlCs and BM-MSCs at all time-points

Conclusion: characteristics of BlCs in vitro appear to be similar to BM-MSCs. Therefore, they could be considered as a substitute for BlCs for a regenerative approach with potential use in future clinical settings for regenerating human appendages

Compare CPM-RMI trial: intramyocardial transplantation of autologous bone marrow-derived cd133+ cells and mononuclear cells during coronary artery bypass grafting in patients with recent myocardial infarction: a phase II/III, multicenter, placebo-controlled, randomized, double-blind clinical trial

Objective: The regenerative potential of bone marrow-derived mononuclear cells [MNCs] and CD133+ stem cells in the heart varies in terms of their pro-angiogenic effects. This phase II/III, multicenter and double-blind trial is designed to compare the functional effects of intramyocardial autologous transplantation of both cell types and placebo in patients with recent myocardial infarction [RMI] post-coronary artery bypass graft

Materials and Methods: This was a phase II/III, randomized, double-blind, placebo-controlled trial COMPARE CPM-RMI [CD133, Placebo, MNCs - recent myocardial infarction] conducted in accordance with the Declaration of Helsinki that assessed the safety and efficacy of CD133 and MNCs compared to placebo in patients with RMI. We randomly assigned 77 eligible RMI patients selected from 5 hospitals to receive CD133+ cells, MNC, or a placebo. Patients underwent gated single photon emission computed tomography assessments at 6 and 18 months post-intramyocardial transplantation. We tested the normally distributed efficacy outcomes with a mixed analysis of variance model that used the entire data set of baseline and between-group comparisons as well as within subject [time] and group×time interaction terms

Results: There were no related serious adverse events reported. The intramyocardial transplantation of both cell types increased left ventricular ejection fraction by 9% [95% confidence intervals [CI]: 2.14% to 15.78%, P=0.01] and improved decreased systolic wall thickening by -3.7 [95% CI: -7.07 to -0.42, P=0.03]. The CD133 group showed significantly decreased non-viable segments by 75% [P=0.001] compared to the placebo and 60% [P=0.01] compared to the MNC group. We observed this improvement at both the 6- and 18-month time points

Conclusion: Intramyocardial injections of CD133+ cells or MNCs appeared to be safe and efficient with superiority of CD133+ cells for patients with RMI. Although the sample size precluded a definitive statement about clinical outcomes, these results have provided the basis for larger studies to confirm definitive evidence about the efficacy of these cell types

Hair follicle generation by injections of adult human follicular epithelial and dermal papilla cells into nude mice

Objective: Dermal papilla and hair epithelial stem cells regulate hair formation and the growth cycle. Damage to or loss of these cells can cause hair loss. Although several studies claim to reconstitute hairs using rodent cells in an animal model, additional research is needed to develop a stable human hair follicle reconstitution protocol. In this study, we have evaluated hair induction by injecting adult cultured human dermal papilla cells and a mixture of hair epithelial and dermal papilla cells in a mouse model

Materials and Methods: In this experimental study, discarded human scalp skins were used to obtain dermal papilla and hair epithelial cells. After separation, cells were cultured and assessed for their characteristics. We randomly allocated 15 C57BL/6 nude mice into three groups that received injections in their dorsal skin. The first group received cultured dermal papilla cells, the second group received a mixture of cultured epithelial and dermal papilla cells, and the third group [control] received a placebo [phosphate-buffered saline [PBS-]]

Results: Histopathologic examination of the injection sites showed evidence of hair growth in samples that received cells compared with the control group. However, the group that received epithelial and dermal papilla cells had visible evidence of hair growth. PKH tracing confirmed the presence of transplanted cells in the new hair

Conclusion: Our data showed that injection of a combination of adult human cultured dermal papilla and epithelial cells could induce hair growth in nude mice. This study emphasized that the combination of human adult cultured dermal papilla and epithelial cells could induce new hair in nude mice

Percutaneous autologous bone marrow-derived mesenchymal stromal cell implantation is safe for reconstruction of human lower limb long bone atrophic nonunion

Objective: Nonunion is defined as a minimum of a 9-month period of time since an injury with no visibly progressive signs of healing for 3 months. Recent studies show that application of mesenchymal stromal cells [MSCs] in the laboratory setting is effective for bone regeneration. Animal studies have shown that MSCs can be used to treat nonunions. For the first time in an Iranian population, the present study investigated the safety of MSC implantation to treat human lower limb long bone nonunion

Materials and Methods: It is a prospective clinical trial for evaluating the safety of using autologus bone marrow derived mesenchymal stromal cells for treating nonunion. Orthopedic surgeons evaluated 12 patients with lower limb long bone nonunion for participation in this study. From these, 5 complied with the eligibility criteria and received MSCs. Under fluoroscopic guidance, patients received a one-time implantation of 20-50x106 MSCs into the nonunion site. All patients were followed by anterior-posterior and lateral X-rays from the affected limb, in addition to hematological, biochemical, and serological laboratory tests obtained before and 1, 3, 6, and 12 months after the implantation. Possible adverse effects that included local or systemic, serious or non-serious, and related or unrelated effects were recorded during this time period

Results: From a safety perspective, all patients tolerated the MSCs implantation during the 12 months of the trial. Three patients had evidence of bony union based on the after implantation X- rays

Conclusion: The results have suggested that implantation of bone marrow-derived MSCs is a safe treatment for nonunion. A double-blind, controlled clinical trial is required to assess the efficacy of this treatment

Mesenchymal stromal cells implantation in combination with platelet lysate product is safe for reconstruction of human long bone nonunion

Objective: nonunion is defined as a minimum of 9 months since injury without any visible progressive signs of healing for 3 months. Recent literature has shown that the application of mesenchymal stromal cells is safe, in vitro and in vivo, for treating long bone nonunion. The present study was performed to investigate the safety of mesenchymal stromal cell [MSC] implantation in combination with platelet lysate [PL] product for treating human long bone nonunion

Materials and Methods: in this case series clinical trial, orthopedic surgeons visited eighteen patients with long bone nonunion, of whom 7 complied with the eligibility criteria. These patients received mesenchymal stromal cells [20 million cells implanted once into the nonunion site using a fluoroscopic guide] in combination with PL product. For evaluation of the effects of this intervention all the patients were followed up by taking anterior-posterior and lateral X-rays of the affected limb before and 1, 3, 6, and 12 months after the implantation. All side effects [local or systemic, serious or non-serious, related or unrelated] were observed during this time period

Results: from a safety perspective the MSC implantation in combination with PL was very well tolerated during the 12 months of the trial. Four patients were healed; based on the control X- ray evidence, bony union had occurred

Conclusion: results from the present study suggest that the implantation of bone marrow-derived MSCs in combination with PL is safe for the treatment of nonunion. A double blind, controlled clinical trial is required to assess the efficacy of this treatment [Registration Number: NCT01206179]

Treatment of hypertrophic scar in human with autologous transplantation of cultured keratinocytes and fibroblasts along with fibrin glue

Hypertrophic scar involves excessive amounts of collagen in dermal layer and may be painful. Nowadays, we can't be sure about effectiveness of procedure for hypertrophic scar management. The application of stem cells with natural scaffold has been the best option for treatment of burn wounds and skin defect, in recent decades. Fibrin glue [FG] was among the first of the natural biomaterials applied to enhance skin deformity in burn patients. This study aimed to identify an efficient, minimally invasive and economical transplantation procedure using novel FG from human cord blood for treatment of hypertrophic scar and regulation collagen synthesis. In this case series study, eight patients were selected with hypertrophic scar due to full-thickness burns. Human keratinocytes and fibroblasts derived from adult skin donors were isolated and cultured. They were tested for the expression of cytokeratin 14 and vimentin using immunocytochemistry. FG was prepared from pooled cord blood. Hypertrophic scars were extensively excised then grafted by simply placing the sheet of FG containing autologous fibroblast and keratinocytes. Histological analyses were performed using Hematoxylin and eosin [H and E] and Masson's Trichrome [MT] staining of the biopsies after 8 weeks. Cultured keratinocytes showed a high level of cytokeratin 14 expression and also fibroblasts showed a high level of vimentin. Histological analyses of skin biopsies after 8 weeks of transplantation revealed re-epithelialization with reduction of hypertrophic scars in 2 patients. These results suggest may be the use of FG from cord blood, which is not more efficient than previous biological transporters and increasing hypertrophic scar relapse, but could lead to decrease pain rate

In vitro differentiation of human umbilical cord blood CD133[+] cells into insulin producing cells in co-culture with rat pancreatic mesenchymal stem cells

Pancreatic stroma plays an important role in the induction of pancreatic cells by the use of close range signaling. In this respect, we presume that pancreatic mesenchymal cells [PMCs] as a fundamental factor of the stromal niche may have an effective role in differentiation of umbilical cord blood cluster of differentiation 133[+] [UCB-CD133[+]] cells into newly-formed beta-cells in vitro. This study is an experimental research. The UCB-CD133[+] cells were purified by magnetic activated cell sorting [MACS] and differentiated into insulin producing cells [IPCs] in co-culture, both directly and indirectly with rat PMCs. Immunocytochemistry and enzyme linked immune sorbent assay [ELISA] were used to determine expression and production of insulin and C-peptide at the protein level. Our results demonstrated that UCB-CD133[+] differentiated into IPCs. Cells in islet-like clusters with [out] co-cultured with rat pancreatic stromal cells produced insulin and C-peptide and released them into the culture medium at the end of the induction protocol. However they did not respond well to glucose challenges. Rat PMCs possibly affect differentiation of UCB-CD133[+] cells into IPCs by increasing the number of immature beta-cells

Differentiation potential of O Bombay human-induced pluripotent stem cells and human embryonic stem cells into fetal erythroid-like cells

There is constant difficulty in obtaining adequate supplies of blood components, as well as disappointing performance of "universal" red blood cells. Advances in somatic cell reprogramming of human-induced pluripotent stem cells [hiPSCs] have provided a valuable alternative source to differentiate into any desired cell type as a therapeutic promise to cure many human disease. In this experimental study, we examined the erythroid differentiation potential of normal Bombay hiPSCs [B-hiPSCs] and compared results to human embryonic stem cell [hESC] lines. Because of lacking ABO blood group expression in B-hiPSCs, it has been highlighted as a valuable source to produce any cell type in vitro. Similar to hESC lines, hemangioblasts derived from B-hiPSCs expressed approximately 9% KDR+CD31+ and approximately 5% CD31+CD34+. In semisolid media, iPSC and hESC-derived hemangioblast formed mixed type of hematopoietic colony. In mixed colonies, erythroid progenitors were capable to express CD71+GPA+HbF+ and accompanied by endothelial cells differentiation. Finally, iPS and ES cells have been directly induced to erythropoiesis without hemangioblast formation that produced CD71+HbF+erythroid cells. Although we observed some variations in the efficiency of hematopoietic differentiation between iPSC and ES cells, the pattern of differentiation was similar among all three tested lines

Fabrication and characterization of spongy denuded amniotic membrane based scaffold for tissue engineering

As a biological tissue material, amniotic membrane [AM] has low immunogenicity and to date has been widely adopted in clinical practice. However, some features such as low biomechanical consistency and rapid biodegradation is limited the application of AM. Therefore, in this study, we fabricated a novel three-dimensional [3D] spongy scaffold made of the extracellular matrix [ECM] of denuded AM. Due to their unique characteristics which are similar to the skin, these scaffolds can be considered as an alternative option in skin tissue engineering. In this experimental study, cellular components of human amniotic membrane [HAM] were removed with 0.03% [w/v] sodium dodecyl sulphate [SDS]. Quantitative analysis was performed to determine levels of Glycosaminoglycans [GAGs], collagen, and deoxyribonucleic acid [DNA]. To increase the low efficiency and purity of the ECM component, especially collagen and GAG, we applied an acid solubilization procedure hydrochloridric acid [HCl 0.1 M] with pepsin [1 mg/ml]. In the present experiment 1-ethyl-3-[3-dimethyl aminopropyl] carbodiimide hydrochloride [EDC]/N-hydroxysuccinimide [NHS] cross linker agent was used to improve the mechanical properties of 3D lyophilized AM scaffold. The spongy 3D AM scaffolds were specified, by scanning electron microscopy, hematoxylin and eosin [H and E] staining, a swelling test, and mechanical strength and in vitro biodegradation tests. Human fetal fibroblast culture systems were used to establish that the scafolds were cytocompatible. Histological analysis of treated human AM showed impressive removal of cellular components. DNA content was diminished after treatment [39 +/- 4.06 micro g/ml vs. 341 +/- 29.60 micro g/ml]. Differences were observed between cellular and denude AM in matrix collagen [478 +/- 18.06 micro g/mg vs. 361 +/- 27.47 micro g/mg].With the optimum concentration of 1 mM NHS/EDC ratio1:4, chemical cross-linker agent could significantly increase the mechanical property, and resistance to collagenase digestion. The results of 2, 4, 6-Trinitrobenzenesulfonic acid [TNBS] test showed that cross-linking efficiency of AM derived ECM scaffolds was about 65% +/- 10.53. Scaffolds treated with NHS/EDC cross-linker agent by 100 micro g/ml collagenase, lost 75% of their dry weight after 14 days. The average pore size of 3D spongy scaffold was 160 micro m measured from scanning electron microscope [SEM] images that it is suitable for cell penetration, nutrients and gas change. In addition, the NHS/ EDC cross-linked AM scaffolds were able to support human fetal fibroblast cell proliferation in vitro. Extracts and contact prepared from the 3D spongy scaffold of AM showed a significant increase in the attachment and proliferation of the human fetal fibroblasts cells. The extra-cellular matrix of denuded AM-based scaffold displays the main properties required for substitute skin including natural in vitro biodegradation, similar physical and mechanical characterization, nontoxic biomaterial and no toxic effect on cell attachment and cell proliferation

Stauprimide priming of human embryonic stem cells toward definitive endoderm

In vitro production of a definitive endoderm [DE] is an important issue in stem cell-related differentiation studies and it can assist with the production of more efficient endoderm derivatives for therapeutic applications. Despite tremendous progress in DE differentiation of human embryonic stem cells [hESCs], researchers have yet to discover universal, efficient and cost-effective protocols. In this experimental study, we have treated hESCs with 200 nM of Stauprimide [Spd] for one day followed by activin A [50 ng/ml; A50] for the next three days [Spd-A50]. In the positive control group, hESCs were treated with Wnt3a [25 ng/ml] and activin A [100 ng/ml] for the first day followed by activin A for the next three days [100 ng/ml; W/A100-A100]. Gene expression analysis showed up regulation of DE-specific marker genes [SOX17, FOXA2 and CXCR4] comparable to that observed in the positive control group. Expression of the other lineage specific markers did not significantly change [p<0.05]. We also obtained the same gene expression results using another hESC line. The use of higher concentrations of Spd [400 and 800 nM] in the Spd-A50 protocol caused an increase in the expression SOX17 as well as a dramatic increase in mortality rate of the hESCs. A lower concentration of activin A [25 ng/ml] was not able to up regulate the DE-specific marker genes. Then, A50 was replaced by inducers of definitive endoderm; IDE1/2 [IDE1 and IDE2], two previously reported small molecule [SM] inducers of DE, in our protocol [Spd-IDE1/2]. This replacement resulted in the up regulation of visceral endoderm [VE] marker [SOX7] but not DE-specific markers. Therefore, while the Spd-A50 protocol led to DE production, we have shown that IDE1/2 could not fully replace activin A in DE induction of hESCs These findings can assist with the design of more efficient chemically-defined protocols for DE induction of hESCs and lead to a better understanding of the different signaling networks that are involved in DE differentiation of hESCs

Five-year follow-up of the local autologous transplantation of CD133+ enriched bone marrow cells in patients with myocardial infarction

The implantation of a CD133+ bone marrow cell population into an ischemic myocardium has emerged as a promising therapeutic modality for myocardial regeneration and restoration of ventricular contractility. While previous studies have documented the short-term safety and efficacy of CD133+ cell transplantation in patients with acute myocardial infarction, there are few reports of long-term follow-up results. Here, we present the results of long-term follow-up of our acute myocardial infarction patients who were treated with intra-myocardial injection of CD133+ cells after coronary bypass graft. After five years, 13 patients in the cell transplantation group and 5 patients in the control group underwent safety and efficacy investigations by New York Heart Association classification and two-dimensional echocardiography [2D echo]. During the five-year study period, no major cardiac adverse events were reported among patients who received CD133+ stem cells. Regarding efficiency, we observed no statistically significant treatment effects for the echocardiographic parameters [left ventricular end-diastolic and end-systolic volumes, and resting ejection fraction] measured during the follow-up period. However, detailed analysis of regional wall motion revealed an improvement in the Wall Motion Score Index from baseline to the six month follow-up, which was maintained during the follow-up period. Taken together, the long-term results of the present study indicate that transplantation of CD133+ is a safe and feasible procedure; however, we could not show any major benefits in our patients. Thus, this issue needs to be addressed by conducting other studies with more patients

Effect of mesenchymal stem cells on doxorubicin-induced fibrosis

The aim of this study was to test the effect of intravenous injection of mesenchymal stem cells [MSCs] on doxorubicin [DOX]-induced fibrosis in the heart. We investigated the mechanisms that possibly mediate this effect. In this experimental study, fibrosis in the myocardium of adult male Wistar rats [weights 180-200 g, 9-10 weeks of age, total n=30] was created by DOX administration. DOX [2.5 mg/kg] was administered intraperitoneally 3 times a week, for a total dose of 15 mg/kg over a period of 2 weeks. MSCs from Wistar rats were separated and cultured in Dulbecco's modified eagle medium [DMEM]. The condition medium [CM] which contained factors secreted by MSCs was also collected from MSCs cultured in serum-free DMEM. Two weeks after the first injection of DOX, MSCs, CM and standard medium [SM] were transplanted via intravenous injection. Four weeks after transplantation, histological [Masson's trichrome staining for fibrosis detection] and molecular [real-time polymerase chain reaction [RT-PCR]] analyses were conducted. In addition, insulin-like growth factor [IGF-1] and hepatocyte growth factor [HGF] in the CM were measured with an enzyme-linked immunosorbent assay [ELISA]. For immunosuppressive treatment, cyclosporine A was given [intraperitoneally, 5 mg/kg/day] starting on the day of surgery until the end of study in all groups. Fibrosis rate and relative gene expression were compared by analysis of variance [ANOVA] and post-Tukey's test. HGF and [IGF-1 in the CM were analyzed by independent sample t test. P<0.01 was considered statistically significant. Our data demonstrated that intravenously transplanted MSCs and CM significantly reduced fibrosis and significantly increased Bcl-2 expression levels in the myocardium compared to the DOX group [p<0.01]. However, there was no significant difference between Bax expression levels in these groups. In addition, secretion of HGF and IGF-1 was detected in the CM [p<0.01]. We conclude that intravenous transplantation of MSCs and CM can attenuate myocardial fibrosis and increase Bcl-2 expression. This may be mediated by paracrine signaling from MSCs via anti-fibrotic and anti-apoptotic factors such as HGF and IGF-1

Surveillance for hepatocellular carcinoma after autologous stem cell transplantation in Cirrhosis

During the resent years there has been interest in using bone marrow stem cells to treat liver cirrhosis. However, there is a potential concern for malignant transformation after stem cell therapy. The aim of this study was to evaluate the development of hepatocellular carcinoma [HCC] after autologous bone marrow stem cell transplantation for liver cirrhosis. All the patients who underwent autologous stem cell transplantation for liver cirrhosis between 2005 and 2011 at our center were enrolled. Cellular infusion was made through peripheral vein, portal vein, or hepatic artery.The patients were invited to undergo screening for hepatocellular carcinoma. The screening was made with ultrasonography and alpha-feto protein [AFP] measurement. Thirty two patients [18 males] were included in the study. Mean age of patients was 45.7 years. Fifteen patients [47%] received mesenchymal stem cell [MSC], 9 [28%] received bone marrow mononuclear cells, 5 [16%] were given CD 133-positive bone marrow cells, and 3 [9%] patients received CD 34-positive bone marrow cells. Mean duration of follow up was 20.5months. Mean serum level of AFP was 2.8 ng/ml at baseline and 3.4ng/ml at the end of follow up [p= 0.3]. One patient was found to have hepatocellular carcinoma three months after infusion of bone marrow mononuclear cells. The incidence rate for HCC was 1.8 cases per 100 person-years in this study. Autologous bone marrow stem cell infusion does not appear to increase the risk of hepatocellular carcinoma. The incidence rate of HCC in this study is comparable or even less than the reported rates of HCC in cohort studies of cirrhotic patients