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Background: Injection of autologous adipose-derived stem cells (ADSCs) and a stromal vascular fraction (VSF) into dermal and subdermal layers promises regenerative advantages by improving skin volume and rejuvenation. Injectable hyaluronic acid (HA) is a temporary dermal filler that, by improving skin hydration, reduces the appearance of fine lines and wrinkles, facial folds and creates structure and volume to the face and lips. This study combined the grafting of micro fragmented fatty tissue with the hyaluronic acid filler procedure, using three different types of HA. Methods: Each sample of micro fragmented adipose tissue harvested using the superficial enhanced fluid fat injection (SEFFI) technique collected from 8 patients were equally divided into two specimens. One of these (EMU specimens) was emulsified by gently applying ten back-and-forth passages from one syringe to another to fluidify the tissue. The other one was not emulsified (Ctrl/NON-EMU specimen). Both EMU and NON-EMU specimens were divided into four aliquots: one served as control, and the others were combined with each of three tested hyaluronic acids. Afterward, we assessed the cellularity of mesenchymal phenotype (defined as the number of adherent cells with mesenchymal phenotype per milliliter of adipose tissue) and the in vitro capacity of differentiation in mesenchymal lineages. Results: Despite low cellularity from emulsified samples combined with HA, isolated cells could grow and expand in culture, thus proving their proliferative ability, showing “good quality” in all conditions (Ctrl/NON-EMU, EMU, and combined with HA). The cells could differentiate towards mesenchymal lineages, express mesenchymal markers by flow cytometry analysis, and maintain their stemness potential. Conclusion: The combination of emulsified harvested tissue with HA products can be exploited to counteract the loss of volume and skin aging of the human face and body. This approach to regenerative aesthetic treatment is a promising treatment for facial antiaging therapy.

Summary Urinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), since they are able to address RNA signatures and atypical mutation identification. Gene expression profiling of DMD individuals’ USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirrors the known transcriptional landscape of DMD muscle and worsens following USCs’ myogenic transformation. This pathogenic transcription signature was reverted by an exon-skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy. The full DMD transcript profile performed in USCs from three undiagnosed DMD individuals addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole-genome sequencing (WGS). This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions, respectively. All three mutations affect DMD gene splicing and cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector) highlighted cell-type variability and the representation of cell-specific DMD isoforms. Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD., We defined urinary stem cell (USC) RNA-seq signatures and provided evidence that they are a non-invasive, affordable cell source to study Duchenne muscular dystrophy (DMD), an X-linked disease, due to dystrophin gene mutations. Based on USCs’ RNA profiling and whole-genome sequencing (WGS), we identified three undetected atypical DMD mutations.

Background New microfat preparations provide material suitable for use as a regenerative filler for different facial areas. To support the development of new robust techniques for regenerative purposes, the cellular content of the sample should be considered. Objectives To evaluate the stromal vascular fraction (SVF) cell components of micro-superficial enhanced fluid fat injection (SEFFI) samples via a technique to harvest re-injectable tissue with minimum manipulation. The results were compared to those obtained from SEFFI samples. Methods Microscopy analysis was performed to visualize the tissue structure. Micro-SEFFI samples were also fractionated using Celector,® an innovative non-invasive separation technique, to provide an initial evaluation of sample fluidity and composition. SVFs obtained from SEFFI and micro-SEFFI were studied. Adipose stromal cells (ASCs) were isolated and characterized by proliferation and differentiation capacity assays. Results Microscopic and quality analyses of micro-SEFFI samples by Celector® confirmed the high fluidity and sample cellular composition in terms of red blood cell contamination, the presence of cell aggregates, and extracellular matrix fragments. ASCs were isolated from adipose tissue harvested using SEFFI and micro-SEFFI systems. These cells were demonstrated to have a good proliferation rate and differentiation potential towards mesenchymal lineages. Conclusions Despite the small sizes and low cellularity observed in micro-SEFFI-derived tissue, we were able to isolate stem cells. This result partially explains the regenerative potential of autologous micro-SEFFI tissue grafts. In addition, using this novel Celector® technology, tissues used for aging treatment were characterized analytically, and the adipose tissue composition was evaluated with no need for extra sample processing. Level of Evidence: 5

Antibiotic resistance is creating enormous attention on the development of new antibiotic-free therapy strategies for bacterial diseases. Mesenchymal stromal stem cells (MSCs) are the most promising candidates in current clinical trials and included in several cell-therapy protocols. Together with the well-known immunomodulatory and regenerative potential of the MSC secretome, these cells have shown direct and indirect anti-bacterial effects. However, the low reproducibility and standardization of MSCs from different sources are the current limitations prior to the purification of cell-free secreted antimicrobial peptides and exosomes. In order to improve MSC characterization, novel label-free functional tests, evaluating the biophysical properties of the cells, will be advantageous for their cell profiling, population sorting, and quality control. We discuss the potential of emerging microfluidic technologies providing new insights into density, shape, and size of live cells, starting from heterogeneous or 3D cultured samples. The prospective application of these technologies to studying MSC populations may contribute to developing new biopharmaceutical strategies with a view to naturally overcoming bacterial defense mechanisms.

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector&reg, (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector&reg, profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine.

Background: Bronchopulmonary dysplasia (BPD) is a chronic lung disease that affects extremely preterm infants and remains – despite improvements in neonatal intensive care – a major cause of neonatal mortality and morbidity. Cell-therapeutic strategies employing mesenchymal stem cells (MSC) have been shown to modulate lung development in BPD models. Objective: Herein, we evaluate the potential of human amniotic fluid (hAF)-SC and hAF-SC with upregulated expression of vascular endothelial growth factor (VEGF) as cell-therapeutic agents for BPD. Methods: Preterm rabbit pups were raised in normoxia (21% O2) or hyperoxia (≥95% O2). Hyperoxia-exposed pups randomly received an intraperitoneal injection of fibroblasts, naïve hAF-SC, or hAF-SC-VEGF on postnatal day (PN) 0. On PN7, surviving pups were tested for pulmonary (forced oscillation technique) and vascular (pulmonary artery Doppler ultrasound) function, and lungs were processed for morphometric measurements of parenchymal and vascular structure and inflammation. Results: Intraperitoneal injection of cells resulted in homing to the lungs. The lungs of hyperoxia-exposed animals displayed parenchymal and vascular structural and functional damage reminiscent of BPD, which was significantly improved after treatment with hAF-SC-VEGF. Treating hyperoxia-exposed animals with naïve AF-SC attenuated only the lung inflammation and the vascular structural defect. Treatment with fibroblasts, which were used as a cellular control, did not lead to any improvements. Conclusion: hAF-SC with upregulated VEGF expression display enhanced potential to prevent/reverse lung injury in preterm rabbits, whereas naïve hAF-SC only show a moderate therapeutic potential. These results point towards an added value of VEGF delivered by hAF-SC in the treatment of BPD.

The use of stem cells for regenerative applications and immunomodulatory effect is increasing. Amniotic epithelial cells (AECs) possess embryonic-like proliferation ability and multipotent differentiation potential. Despite the simple isolation procedure, inter-individual variability and different isolation steps can cause differences in isolation yield and cell proliferation ability, compromising reproducibility observations among centers and further applications. We investigated the use of a new technology as a diagnostic tool for quality control on stem cell isolation. The instrument label-free separates cells based on their physical characteristics and, thanks to a micro-camera, generates a live fractogram, the fingerprint of the sample. Eight amniotic membranes were processed by trypsin enzymatic treatment and immediately analysed. Two types of profile were generated: a monomodal and a bimodal curve. The first one represented the unsuccessful isolation with all recovered cell not attaching to the plate, while for the second type, the isolation process was successful, but we discovered that only cells in the second peak were alive and resulted adherent. We optimized a Quality Control (QC) method to define the success of AEC isolation using the fractogram generated. This predictive outcome is an interesting tool for laboratories and cell banks that isolate and cryopreserve fetal annex stem cells for research and future clinical applications.

Objective: Glucagon-like peptide-1 (GLP-1) increases surfactant protein expression in type 2 pneumocytes. Herein, we determine if transplacental GLP-1 treatment accelerates lung growth in the fetal rabbit model of congenital diaphragmatic hernia (DH). Methods: Time-mated does had an induction of DH on day 23 followed by daily GLP-1 or placebo injection until term. At that time, the does were weighed, fetal blood was obtained for GLP-1 assay, and the lungs were dissected. Fetal outcome measures were lung-to-body-weight ratio (LBWR), morphometry, and Ki67 and surfactant protein B (SPB) expression. Results: Maternal weight loss in the GLP-1 group was 7.1%. Fetal survival was lower in GLP-1 fetuses compared to placebo controls (27/85, 32% vs. 35/57, 61%; p < 0.05). Fetal GLP-1 levels were increased 3.6-fold. The LBWR of GLP-1 DH fetuses fell within the range of DH placebo fetuses (1.166 ± 0.207% vs. 1.312 ± 0.418%), being significantly lower than that of placebo-exposed unoperated fetuses (2.280 ± 0.522%; p < 0.001). GLP-1 did not improve airway morphometry. GLP-1 DH lungs had a reduced adventitial and medial thickness within the range of controls, and lesser muscularization of vessels measuring 30-60 µm. There were no differences in Ki67 and SPB expression. Conclusion: GLP-1 at this dosage improves peripheric pulmonary vessel morphology in intra-acinar vessels with no effect on airway morphometry but with significant maternal and fetal side effects. Thus, it is an unlikely medical strategy.

What's already known about this topic? Stem cells can be isolated from amniotic fluid and may be used for cell therapy and tissue engineering applications in perinatal medicine. Clinical implementation requires development of an adapted cell banking system including optimal processing and cryopreservation of amniotic fluid samples, with maintenance of cell viability and characteristics after thawing. What does this study add? We identified three suitable cryopreservation protocols of fresh amniotic fluid samples with high cell recovery rate and unchanged stem cell characteristics post-thawing. One of these is compatible with current good manufacturing practice legislation and can be performed in fetal medicine units without stem cell culture expertise. Objectives: Stem cells (SCs) can be isolated from amniotic fluid (AF) for a variety of perinatal applications. In view of this, we compared different cryopreservation protocols for these AFSCs. Methods: We screened seven freezing and thawing protocols using two well-established human AFSC lines: freezing protocol 1 (FP1), 10% dimethyl sulfoxide (DMSO); FP2, 2.5% DMSO, caspase inhibitor, and catalase; FP3, 5% glycerol, caspase inhibitor, and catalase; FP4, sperm freezing medium; FP5, slow-freezing solution; FP6, ethylene glycol, sucrose, and Ficoll 70; and FP7, vitrification solution. Outcome measures were post-thawing cell viability, recovery, doubling time and mesenchymal SC markers. The three best performing protocols were subsequently tested on cells isolated from clinical consecutive freshly harvested AF samples from two fetal medicine units. Results: Protocols 1, 5, and 6 performed significantly better on well-characterized cell lines. They performed equally well on cell pellets from freshly harvested AF (n=28). Conclusions: We identified three suitable cryopreservation protocols because of high cell recovery and unchanged SC characteristics. Given one of these, the slow-freezing solution, is compatible with current good manufacturing practice legislation, it may be ultimately clinically used.

Purpose The reconstruction of congenital diaphragmatic hernia or other congenital soft tissue defects often requires implants. These can be either degradable or permanent, each having their advantages. Whatever type is being used, the host response induced by implants plays a crucial role to determine the outcome. Macrophages are pivotal during implant remodeling; they are plastic and acquire in response to environmental stimuli either an inflammatory status and mediate subsequent fibrosis or a regulatory status and facilitate functional remodeling. Matrices engineered with mesenchymal stem cells (MSCs) have the capacity to modulate the host immune reaction. MSCs are believed to promote constructive remodeling of the implant through a regulatory macrophage response among others. Herein, we evaluate this potential of MSC derived from the amniotic fluid (AF-MSC), an interesting MSC type for neonatal reconstruction, on electrospun polylactic acid (PLA) scaffolds. Methods We seeded AF-MSC at a density of 1.105/cm2 on electrospun PLA matrices and determined cell viability. In vivo, we used cell-seeded or cell-free PLA matrices for subcutaneous implantation in immune competent rats. The host immune response was evaluated by histomorphometry at 14 days postoperatively. Results The PLA matrix supported adherence and proliferation of AF-MSC. Fourteen days after implantation, PLA matrices were well penetrated by inflammatory cells, new blood vessels, and collagen fibers. AF-MSC–seeded scaffolds were associated with a similar response yet with a decreased number of eosinophils, increased matrix degradation and collagen fiber deposition compared with controls. The amount of total macrophages and of M2-subtype was similar for all animals. Conclusion Electrospun PLA matrices are a suitable substrate for short-term culture of AF-MSC. In rats, addition of AF-MSC to PLA matrices modulates the host response after subcutaneous implantation, yet without a difference in macrophage profile compared with control.

The amniotic fluid is a clinically accessible source of mesenchymal stem cells (AF-MSC) during gestation, which enables autologous cellular therapy for perinatal disorders. The origin of AF-MSC remains elusive: renal and neuronal progenitors have been isolated from the AF-MSC pool, yet no cells with pulmonary characteristics. We analyzed gene expression of pulmonary and renal markers of 212 clonal lines of AF-MSC isolated from amniocentesis samples. AF-MSC were cultured on dishes coated with extracellular matrix (ECM) proteins from decellularized fetal rabbit lungs. In vivo differentiation potential of AF-MSC that expressed markers suggestive of lung fate was tested by renal subcapsular injections in immunodeficient mice. Of all the isolated AF-MSC lines, 26% were positive for lung endodermal markers FOXA2 and NKX2.1 and lacked expression of renal markers (KSP). This AF-MSC subpopulation expressed other lung-specific factors, including IRX1, P63, FOXP2, LGR6, SFTC, and PDPN. Pulmonary marker expression decreased over passages when AF-MSC were cultured under conventional conditions, yet remained more stable when culturing the cells on lung ECM-coated dishes. Renal subcapsular injection of AF-MSC expressing lung-specific markers resulted in engrafted cells that were SPTB positive. These data suggest that FOXA2+/NKX2.1+/KSP- AF-MSC lines have lung characteristics which are supported by culture on lung ECM-coated dishes.

Introduction Stem cells (SCs) isolated from amniotic fluid (AF) are a promising source for autologous perinatal cell therapy. The aim of this study was to develop a routine isolation, selection, and expansion protocol of clonal SC lines from redundant clinical amniocentesis samples. Materials and methods Amniotic fluids were collected between 15 and 22 weeks of gestation, and SCs were isolated by CD117-based and mechanical selection protocols. SCs were characterized by mesenchymal SC marker expression and differentiation protocols. Cells were manipulated with a lentiviral vector system expressing the β-galactosidase reporter gene and were injected into immunodeficient newborn mouse pups. Qualitative assessment was performed to detect the infused cells after 1 week. Results A total of 78 clonal AF SC populations were successfully isolated by mechanical selection from 21 consecutive amniocentesis samples. They were positive for mesenchymal SC cluster of differentiation markers and could be differentiated into the different lineages. SCs were stably labeled using β-galactosidase and were detected in the lungs and hearts of the neonatal mice. Conclusion We demonstrate that mesenchymal SCs can be routinely isolated and clonally expanded from mid-gestation human AF using mechanical isolation. They can easily be transduced and be tested for perinatal treatment in animal models. © 2013 John Wiley & Sons, Ltd.

Introduction Both embryonic and adult tissues are sources of stem cells with therapeutic potential but with some limitations in the clinical practice such as ethical considerations, difficulty in obtaining and tumorigenicity. As an alternative, the placenta is a foetal tissue that can be obtained during gestation and at term, and it represents a reservoir of stem cells with various potential. Sources of data We reviewed the relevant literature concerning the main stem cells that populate the placenta. Areas of agreement Recently, the placenta has become useful source of stem cells that offer advantages in terms of proliferation and plasticity when compared with adult cells and permit to overcome the ethical and safety concern inherent in embryonic stem cells. In addition, the placenta has the advantage of containing epithelia, haematopoietic and mesenchymal stem cells. These stem cells possess immunosuppressive properties and have the capacity of suppress in vivo inflammatory responses. Areas of controversy Some studies describe a subpopulation of placenta stem cells expressing pluripotency markers, but for other studies, it is not clear whether pluripotent stem cells are present during gestation beyond the first few weeks. Particularly, the expression of some pluripotency markers such as SSEA-3, TRA-1-60 and TRA-1-81 has been reported by us, but not by others. Growing points Placenta stem cells could be of great importance after delivery for banking for autologous and allogeneic applications. The beneficial effects of these cells may be due to secretion of bioactive molecules that act through paracrine actions promoting beneficial effects. Areas timely for developing research Understanding the role of placenta stem cells during pregnancy and their paracrine actions could help in the study of some diseases that affect the placenta during pregnancy.

OBJECTIVE: To evaluate if the up-regulation of vascular endothelial growth factor strengthens the protective effect of amniotic fluid stem cells in a renal ischemia-reperfusion injury model. DESIGN: Randomized animal study. SETTINGS: University research laboratory. SUBJECTS: A total of 40 males 12-week-old Wistar rats were subjected to ischemia-reperfusion and assigned to four groups: amniotic fluid stem cells, vascular endothelial growth factor-amniotic fluid stem cells in two different doses, and vehicle. Ten animals were used as sham-controls. INTERVENTION: Six hours after induction of renal ischemia-reperfusion injury, amniotic fluid stem cells, vascular endothelial growth factor-amniotic fluid stem cells in two different doses, or vehicle were injected intraarterially. MEASUREMENTS AND MAIN RESULTS: Analyses were performed at 24 hours, 48 hours, and 2 months after treatment. Outcome measures included serum creatinine, urine microprotenuira, and immunohistomorphometric analyses. Vascular endothelial growth factor-amniotic fluid stem cells induced a significantly higher nephroprotection than amniotic fluid stem cells. This effect was mediated mainly by immunomodulation, which led to lower macrophage infiltration and higher presence of regulatory T cell after ischemia-reperfusion injury. At medium term, it inhibited the progression toward chronic kidney disease. Vascular endothelial growth factor-amniotic fluid stem cells can worsen the ischemia-reperfusion injury when delivered in a high dose. CONCLUSIONS: Up-regulation of vascular endothelial growth factor enhances the therapeutic effect of human amniotic fluid stem cells in rats with renal ischemia-reperfusion injury, mainly by mitogenic, angiogenic, and anti-inflammatory mechanisms. ispartof: Crit Care Med vol:45 issue:1 pages:e86-e96 ispartof: location:United States status: published

Amniotic fluid stem cells (AFSc) are a very heterogeneous subtype of stem cells with a broad multi potential. They could be used to treat congenital malformations or diseases. Recently, mesoangioblasts, resident pericytes of skeletal muscles, were shown to undergo muscle differentiation in vitro and in vivo. In this study we focused on the identification of an AFS subtype with pericytic characteristics and evaluate its myogenic potential. We identified monoclonal AFSc lines expressing alkaline phosphatase activity (ALP) and the canonical pericytic markers neural-glial-2 chondroitin sulphate proteglycan (NG2), platelet derived growth factor receptor α and β (PDGFR-α, -β) and α smooth muscle actin (α-SMA). These cells were able to integrate into the newly formed myotubes when co-cultured with the C2C12 cells. To test the paracrine effects of these AFSC on muscle regeneration, we assessed their affects in a transwell assay with acutely injured myotubes. AFSc were able to modulate the expression of specific growth factors involved in muscle regeneration, such as Transforming Growth Factor β (Tgfβ), Interferon γ (Ifnγ), Hepatocyte Growth Factor (Hgf) and Matrix Metalloproteinase 2 (Mmp2). When AFSc were injected in injured muscles they ameliorated muscle repair as measured by the reduction of centronucleated fibers and fibrosis. Interestingly, the transcriptional program of growth factor response in vitro is observed in large part in the in vivo xenograft experimental model, with the extension of Myostatin and Matrix Metalloproteinase 9 (Mmp9). Our data suggest that AFSc subtype with pericytic characteristics have the ability to modulate muscle regeneration in vitro and in vivo. ispartof: Journal of Stem Cell Research & Therapy vol:6 status: published

In humans, nephrogenesis is completed prenatally, with nephrons formed until 34 weeks of gestational age. We hypothesized that urine of preterm neonates born before the completion of nephrogenesis is a noninvasive source of highly potent stem/progenitor cells. To test this hypothesis, we collected freshly voided urine at day 1 after birth from neonates born at 31-36 weeks of gestational age and characterized isolated cells using a single-cell RT-PCR strategy for gene expression analysis and flow cytometry and immunofluorescence for protein expression analysis. Neonatal stem/progenitor cells expressed markers of nephron progenitors but also, stromal progenitors, with many single cells coexpressing these markers. Furthermore, these cells presented mesenchymal stem cell features and protected cocultured tubule cells from cisplatin-induced apoptosis. Podocytes differentiated from the neonatal stem/progenitor cells showed upregulation of podocyte-specific genes and proteins, albumin endocytosis, and calcium influx via podocyte-specific transient receptor potential cation channel, subfamily C, member 6. Differentiated proximal tubule cells showed upregulation of specific genes and significantly elevated p-glycoprotein activity. We conclude that urine of preterm neonates is a novel noninvasive source of kidney progenitors that are capable of differentiation into mature kidney cells and have high potential for regenerative kidney repair.

OBJECTIVE: With the increased use of invasive fetal procedures, the number of patients facing postprocedure membrane rupture is increasing. We aimed to describe the use of platelets and fresh frozen plasma for sealing iatrogenic fetal membrane defects. PATIENTS AND METHODS: We describe the mechanisms of action of the amniopatch procedure as well as published experience. RESULTS: Amniopatch effectively sealed the fetal membranes in over two thirds of published cases (n=44). There is a risk of 17% of in utero fetal death, which may occur remotely from the procedure and is often unexplained. DISCUSSION AND CONCLUSION: In case of early onset but persistent amniotic fluid leakage following an invasive fetal procedure, amniopatch may be offered.

OBJECTIVES: Mesenchymal stem cells derived from human amniotic fluid (hAFSCs) are a promising source for cellular therapy, especially for renal disorders, as a subpopulation is derived from the fetal urinary tract. The purpose of this study was to evaluate if hAFSCs with a renal progenitor phenotype demonstrate a nephroprotective effect in acute ischemia reperfusion (I/R) model and prevent late stage fibrosis. METHODS: A total of 45 male 12-wk-old Wistar rats were divided into three equal groups;: rats subjected to I/R injury and treated with Chang Medium, rats subjected to I/R injury and treated with hAFSCs and sham-operated animals. In the first part of this study, hAFSCs that highly expressed CD24, CD117, SIX2 and PAX2 were isolated and characterized. In the second part, renal I/R injury was induced in male rats and cellular treatment was performed 6 hours later via arterial injection. Functional and histological analyses were performed 24 hours, 48 hours and 2 months after treatment using serum creatinine, urine protein to creatinine ratio, inflammatory and regeneration markers and histomorphometric analysis of the kidney. Statistical analysis was performed by analysis of variance followed by the Tukey's test for multiple comparisons or by nonparametric Kruskal-Wallis followed by Dunn. Statistical significance level was defined as p

Stem cells (SCs) can be isolated from amniotic fluid (AF) for a variety of perinatal applications. In view of this, we compared different cryopreservation protocols for these AFSCs.We screened seven freezing and thawing protocols using two well-established human AFSC lines: freezing protocol 1 (FP1), 10% dimethyl sulfoxide (DMSO); FP2, 2.5% DMSO, caspase inhibitor, and catalase; FP3, 5% glycerol, caspase inhibitor, and catalase; FP4, sperm freezing medium; FP5, slow-freezing solution; FP6, ethylene glycol, sucrose, and Ficoll 70; and FP7, vitrification solution. Outcome measures were post-thawing cell viability, recovery, doubling time and mesenchymal SC markers. The three best performing protocols were subsequently tested on cells isolated from clinical consecutive freshly harvested AF samples from two fetal medicine units.Protocols 1, 5, and 6 performed significantly better on well-characterized cell lines. They performed equally well on cell pellets from freshly harvested AF (n = 28).We identified three suitable cryopreservation protocols because of high cell recovery and unchanged SC characteristics. Given one of these, the slow-freezing solution, is compatible with current good manufacturing practice legislation, it may be ultimately clinically used.

In the EU-27, 2,100 babies with congenital diaphragmatic hernia (CDH) are born annually. CDH is fatal in 30% of them. Experimental fetal surgery in severe cases results in a survival rate of 50 to 60% at its best. Failure is due to insufficient lung growth, persistent pulmonary hypertension or prematurity induced by the procedure. For nonsurvivors alternative strategies are required. Survivors undergo anatomical repair, but large diaphragmatic defects are closed using a patch. At present the used materials are less than ideal, mainly because of recurrence and chest deformation. To overcome the above limitations, alternative medical therapies (pharmacologic or cell therapy) that are more potent and less invasive are needed. Also a more functional postnatal repair may be possible when using novel scaffolds or engineered constructs. We see a prominent place for autologous amniotic fluid-derived stem cells for these novel strategies, which could be prenatally harvested following appropriate patient selection by noninvasive imaging.

Purpose. To investigatein vivothe acute host response to an alternative implant designed for the treatment of stress urinary incontinence (SUI) and pelvic organ prolapse (POP).Methods. A biodegradable scaffold was produced from poly-L-lactic acid (PLA) using the electrospinning technique. Human and rat adipose-derived stem cells (ADSCs) were isolated and characterized by fluorescence-activated cell sorting and differentiation assays. PLA scaffolds were seeded and cultured for 2 weeks with human or rat ADSCs. Scaffolds with and without human or rat ADSCs were implanted subcutaneously on the abdominal wall of rats. After 3 and 7 days, 6 animals from each group were sacrificed. Sections from each sample were analyzed by Haematoxylin and Eosin staining, Sirius red staining, and immunohistochemistry for CD68, PECAM-1, and collagen I and III.Results. Animals responded to the scaffolds with an acute macrophage response. After 7 days of implantation, there was extensive host cell penetration, new blood vessel formation, and new collagen deposition throughout the full thickness of the samples without obvious differences between cell-containing and cell-free scaffolds.Conclusions. The acutein vivoresponse to an alternative implant (both with and without cells) for the treatment of SUI and POP showed good acute integration into the host tissues.

Intra-arterial injection of mesenchymal stem cells has been proven to result in a superior nephroprotection compared to intravenous injection. This avoids initial passage through filter organs such as the lung, liver and spleen. The aim of the present study was to investigate whether suprarenal aortic delivery results in a homogenous distribution to both kidneys. Chinese ink was used to evaluate the renal distribution pattern for the comparison of two retrograde intra-aortic injection methods. In the first, the aorta caudal to the renal branches was temporarily clamped and Chinese ink was injected at the level of the renal arteries. In the second, a distal aortic clamp was combined with alternated clamping of the contralateral arteries. Immediately after injection, kidneys were harvested for histological analysis. Amniotic fluid stem cells labeled with LacZ were injected in the aorta by alternated clamping of the renal arteries in order to track the cells in a rat ischemia/reperfusion model. Without renal artery clamping, intra-aortic administration resulted in a delivery of the ink into the right kidney, whereas administration with alternated clamping of the contralateral renal artery, together with distal aortic artery clamping, resulted in a more homogenous distribution of the ink in both kidneys. Moreover, LacZ-positive cells were found in both kidneys after 6 h of injection. In conclusion, the retrograde administration of Chinese ink in two steps is a fast and reproducible technique, which results in a more homogenous distribution of the stain in both kidneys than a single administration combined by only clamping the aorta.