Silvia Zia

Placenta as a reservoir of stem cells: an underutilized resource?

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.

Routine clonal expansion of mesenchymal stem cells derived from amniotic fluid for perinatal applications

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.

Amniotic Fluid Derived Stem Cells with a Renal Progenitor Phenotype Inhibit Interstitial Fibrosis in Renal Ischemia and Reperfusion Injury in Rats

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 <0.05. Results hAFSCs treatment resulted in significantly reduced serum creatinine level at 24 hours, less tubular necrosis, less hyaline cast formation, higher proliferation index, less inflammatory cell infiltration and less myofibroblasts at 48h. The treated group had less fibrosis and proteinuria at 2 months after injury. Conclusion hAFSCs contain a renal progenitor cell subpopulation that has a nephroprotective effect when delivered intra-arterially in rats with renal I/R injury, and reduces interstitial fibrosis on long term follow-up.

Acute In Vivo Response to an Alternative Implant for Urogynecology

Purpose. To investigate in vivo the 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 acute in vivo response to an alternative implant (both with and without cells) for the treatment of SUI and POP showed good acute integration into the host tissues.

Safe and effective cryopreservation methods for long-term storage of human-amniotic-fluid-derived stem cells

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.

Routine isolation and expansion late mid trimester amniotic fluid derived mesenchymal stem cells in a cohort of fetuses with congenital diaphragmatic hernia

OBJECTIVE To assess the feasibility of routine isolation and expansion of amniotic fluid derived mesenchymal stem cells (AF-MSC) in fetuses diagnosed with isolated congenital diaphragmatic hernia (CDH). STUDY DESIGN Redundant AF samples of fetuses with CDH and normal fetuses were obtained. Cell colonies were mechanically selected for each sample. Proliferation capacity was expressed as population doubling time (PDT). Cell lines were further characterized with flow cytometry, differentiation assays and qRT-PCR (OCT4 and NANOG). After cell labeling with LacZ in vivo tracking was performed after fetal tracheal injection in rabbits. RESULTS Fourteen consecutive CDH samples (median gestational age (GA) of 32.9 weeks; IQR: 27.8-34.3 weeks) and seven control samples (30 weeks; IQR: 28.9-34.4 weeks) were obtained. PDT was similar in both groups (45.4h±1.9 vs. 52.3h±3.4;NS). AF-MSCs expressed a typical mesenchymal CD marker profile. Clones could be differentiated in osteogenic, adipogenic and chrondrogenic lineages. Expression of multipotency markers was low in all cell lines. We confirmed the presence of injected cells inside the fetal lung three days after intratracheal injection. CONCLUSION Routine isolation and expansion of AF-MSCs in CDH is feasible and cell lines generated were comparable to those of control samples. AF-MSCs from affected fetuses could potentially be used in future stem cell therapy.

Urine of Preterm Neonates as a Novel Source of Kidney Progenitor Cells

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.

Medical and Regenerative Solutions for Congenital Diaphragmatic Hernia: A Perinatal Perspective

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.

Vascular Endothelial Growth Factor Up-regulation in Human Amniotic Fluid Stem Cell Enhances Nephroprotection After Ischemia-Reperfusion Injury in the Rat

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.

The Effect of Transplacental Administration of Glucagon-Like Peptide-1 on Fetal Lung Development in the Rabbit Model of Congenital Diaphragmatic Hernia

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.