Laura Perin

Human Amniotic Fluid Stem Cells Can Integrate and Differentiate into Epithelial Lung Lineages

A new source of stem cells has recently been isolated from amniotic fluid; these amniotic fluid stem cells have significant potential for regenerative medicine. These cells are multipotent, showing the ability to differentiate into cell types from each embryonic germ layer. We investigated the ability of human amniotic fluid stem cells (hAFSC) to integrate into murine lung and to differentiate into pulmonary lineages after injury. Using microinjection into cultured mouse embryonic lungs, hAFSC can integrate into the epithelium and express the early human differentiation marker thyroid transcription factor 1 (TTF1). In adult nude mice, following hyperoxia injury, tail vein‐injected hAFSC localized in the distal lung and expressed both TTF1 and the type II pneumocyte marker surfactant protein C. Specific damage of Clara cells through naphthalene injury produced integration and differentiation of hAFSC at the bronchioalveolar and bronchial positions with expression of the specific Clara cell 10‐kDa protein. These results illustrate the plasticity of hAFSC to respond in different ways to different types of lung damage by expressing specific alveolar versus bronchiolar epithelial cell lineage markers, depending on the type of injury to recipient lung.

Renal differentiation of amniotic fluid stem cells

Abstract.  Objectives: The role of stem cells in regenerative medicine is evolving rapidly. Here, we describe the application, for kidney regeneration, of a novel non‐genetically modified stem cell, derived from human amniotic fluid. We show that these pluripotent cells can develop and differentiate into de novo kidney structures during organogenesis in vitro. Materials and methods: Human amniotic fluid‐derived stem cells (hAFSCs) were isolated from human male amniotic fluid obtained between 12 and 18 weeks gestation. Green fluorescent protein and Lac‐Z‐transfected hAFSCs were microinjected into murine embryonic kidneys (12.5–18 days gestation) and were maintained in a special co‐culture system in vitro for 10 days. Techniques of live microscopy, histology, chromogenic in situ hybridization and reverse transcriptase polymerase chain reaction were used to characterize the hAFSCs during their integration and differentiation in concert with the growing organ. Results: Green fluorescent protein and Lac‐Z‐transfected hAFSCs demonstrated long‐term viability in organ culture. Histological analysis of injected kidneys revealed that hAFSCs were capable of contributing to the development of primordial kidney structures including renal vesicle, C‐ and S‐shaped bodies. Reverse transcriptase polymerase chain reaction confirmed expression of early kidney markers for: zona occludens‐1, glial‐derived neurotrophic factor and claudin. Conclusions: Human amniotic fluid‐derived stem cells may represent a potentially limitless source of ethically neutral, unmodified pluripotential cells for kidney regeneration.

A Periodic Diet that Mimics Fasting Promotes Multi-System Regeneration, Enhanced Cognitive Performance, and Healthspan

Prolonged fasting (PF) promotes stress resistance, but its effects on longevity are poorly understood. We show that alternating PF and nutrient-rich medium extended yeast lifespan independently of established pro-longevity genes. In mice, 4 days of a diet that mimics fasting (FMD), developed to minimize the burden of PF, decreased the size of multiple organs/systems, an effect followed upon re-feeding by an elevated number of progenitor and stem cells and regeneration. Bi-monthly FMD cycles started at middle age extended longevity, lowered visceral fat, reduced cancer incidence and skin lesions, rejuvenated the immune system, and retarded bone mineral density loss. In old mice, FMD cycles promoted hippocampal neurogenesis, lowered IGF-1 levels and PKA activity, elevated NeuroD1, and improved cognitive performance. In a pilot clinical trial, three FMD cycles decreased risk factors/biomarkers for aging, diabetes, cardiovascular disease, and cancer without major adverse effects, providing support for the use of FMDs to promote healthspan.

Protective effect of human amniotic fluid stem cells in an immunodeficient mouse model of acute tubular necrosis

Acute Tubular Necrosis (ATN) causes severe damage to the kidney epithelial tubular cells and is often associated with severe renal dysfunction. Stem-cell based therapies may provide alternative approaches to treating of ATN. We have previously shown that clonal c-kitpos stem cells, derived from human amniotic fluid (hAFSC) can be induced to a renal fate in an ex-vivo system. Herein, we show for the first time the successful therapeutic application of hAFSC in a mouse model with glycerol-induced rhabdomyolysis and ATN. When injected into the damaged kidney, luciferase-labeled hAFSC can be tracked using bioluminescence. Moreover, we show that hAFSC provide a protective effect, ameliorating ATN in the acute injury phase as reflected by decreased creatinine and BUN blood levels and by a decrease in the number of damaged tubules and apoptosis therein, as well as by promoting proliferation of tubular epithelial cells. We show significant immunomodulatory effects of hAFSC, over the course of ATN. We therefore speculate that AFSC could represent a novel source of stem cells that may function to modulate the kidney immune milieu in renal failure caused by ATN.

Prolonged Fasting Reduces IGF-1/PKA to Promote Hematopoietic-Stem-Cell-Based Regeneration and Reverse Immunosuppression

Immune system defects are at the center of aging and a range of diseases. Here, we show that prolonged fasting reduces circulating IGF-1 levels and PKA activity in various cell populations, leading to signal transduction changes in long-term hematopoietic stem cells (LT-HSCs) and niche cells that promote stress resistance, self-renewal, and lineage-balanced regeneration. Multiple cycles of fasting abated the immunosuppression and mortality caused by chemotherapy and reversed age-dependent myeloid-bias in mice, in agreement with preliminary data on the protection of lymphocytes from chemotoxicity in fasting patients. The proregenerative effects of fasting on stem cells were recapitulated by deficiencies in either IGF-1 or PKA and blunted by exogenous IGF-1. These findings link the reduced levels of IGF-1 caused by fasting to PKA signaling and establish their crucial role in regulating hematopoietic stem cell protection, self-renewal, and regeneration.

Stem/Progenitor Cells in Lung Development, Injury Repair, and Regeneration

At least two populations of epithelial stem/progenitor cells give rise to the lung anlage, comprising the laryngo-tracheal complex versus the distal lung below the first bronchial bifurcation. Amplification of the distal population requires FGF9-FGF10-FGFR2b-Sprouty signaling. Residual pools of adult stem cells are hypothesized to be the source of lung regeneration and repair. These pools have been located within the basal layer of the upper airways, within or near pulmonary neuroendocrine cell rests, at the bronchoalveolar junction as well as within the alveolar epithelial surface. Rapid repair of the denuded alveolar surface after injury is clearly key to survival. Strategies to enhance endogenous alveolar epithelial repair could include protection of epithelial progenitors from injury and/or stimulation of endogenous progenitor cell function. Protection with inosine or FGF signaling are possible small molecule therapeutic options. Alternatively, exogenous stem/progenitor cells can be delivered into the lung either intravenously, intratracheally, or by direct injection. Sources of exogenous stem/progenitor cells that are currently under evaluation in the context of acute lung injury repair include embryonic stem cells, bone marrow- or fat-derived mesenchymal stem cells, circulating endothelial progenitors, and, recently, amniotic fluid stem/progenitor cells. Further work will be needed to translate stem/progenitor cell therapy for the lung.

Human Amniotic Fluid as a Potential New Source of Organ Specific Precursor Cells for Future Regenerative Medicine Applications

PURPOSE Human amniotic fluid contains multiple cell types, including pluripotent and committed progenitor cells, and fully differentiated cells. We characterized various cell populations in amniotic fluid. MATERIALS AND METHODS Optimum culture techniques for multiple cell line passages with minimal morphological change were established. Cell line analysis and characterization were done with reverse transcriptase and real-time polymerase chain reaction. Immunoseparation was done to distinguish native progenitor cell lines and their various subpopulations. RESULTS Endodermal and mesodermal marker expression was greatest in samples of early gestational age while ectodermal markers showed a constant rate across all samples. Pluripotent and mesenchymal cells were always present but hematopoietic cell markers were expressed only in older samples. Specific markers for lung, kidney, liver and heart progenitor cells were increasingly expressed after 18 weeks of gestation. We specifically focused on a CD24+OB-cadherin+ population that could identify uninduced metanephric mesenchyma-like cells, which in vivo are nephron precursors. The CD24+OB-cadherin+ cell line was isolated and subjected to further immunoseparation to select 5 distinct amniotic fluid kidney progenitor cell subpopulations based on E-cadherin, podocalyxin, nephrin, TRKA and PDGFRA expression, respectively. CONCLUSIONS These subpopulations may represent different precursor cell lineages committed to specific renal cell fates. Committed progenitor cells in amniotic fluid may provide an important and novel resource of useful cells for regenerative medicine purposes.

Characterization of Human Amniotic Fluid Stem Cells and Their Pluripotential Capability

Over the past decade, there has been ever-increasing emphasis placed on stem cells and their potential role in regenerative medicine for reconstruction of bio-artificial tissues and organs. Scientists have looked at various sources for pluripotential cells ranging from embryonic stem cells to adult stem cells. Amniocentesis is a well-established technique for the collection of cells derived from the human embryo. In this chapter, we are going to describe how to isolate, maintain in culture, and characterize the pluripotential capabilities of stem cells derived from amniocentesis in an in vitro and in vivo system. Cell samples are obtained from human pregnancies, and the progenitor cells are isolated from male fetuses with a normal karyotype in order to confirm the absence of maternal admixed cells. Progenitor cells express embryonic-specific cell markers, they show a high self-renewal capacity with 350 population doublings, and normal ploidy is confirmed by cell-cycle analyses. They maintain their undifferentiated state, pluripotential ability, clonogenicity, and telomere length over the population doublings. The progenitor cells are inducible to different cell lineages (osteogenic, adipogenic, skeletal muscle, endothelial, neuronal, and hepatic cells) under specific growth conditions. The ability to induce cell-type-specific differentiation is confirmed by phenotypic changes, immunocytochemistry, gene expression, and functional analyses. In addition, we will describe an application of these cells in an ex vivo and in vivo system for potential in organ (renal) regeneration. The progenitor cells described in this chapter have a high potential for expansion, and may be a good source for research and therapeutic applications where large numbers of cells are needed. Progenitor cells isolated during gestation may be beneficial for fetuses diagnosed with malformations and could be cryopreserved for future self-use.

Injection of Amniotic Fluid Stem Cells Delays Progression of Renal Fibrosis

Injection of amniotic fluid stem cells ameliorates the acute phase of acute tubular necrosis in animals by promoting proliferation of injured tubular cells and decreasing apoptosis, but whether these stem cells could be of benefit in CKD is unknown. Here, we used a mouse model of Alport syndrome, Col4a5(-/-) mice, to determine whether amniotic fluid stem cells could modify the course of progressive renal fibrosis. Intracardiac administration of amniotic fluid stem cells before the onset of proteinuria delayed interstitial fibrosis and progression of glomerular sclerosis, prolonged animal survival, and ameliorated the decline in kidney function. Treated animals exhibited decreased recruitment and activation of M1-type macrophages and a higher proportion of M2-type macrophages, which promote tissue remodeling. Amniotic fluid stem cells did not differentiate into podocyte-like cells and did not stimulate production of the collagen IVa5 needed for normal formation and function of the glomerular basement membrane. Instead, the mechanism of renal protection was probably the paracrine/endocrine modulation of both profibrotic cytokine expression and recruitment of macrophages to the interstitial space. Furthermore, injected mice retained a normal number of podocytes and had better integrity of the glomerular basement membrane compared with untreated Col4a5(-/-) mice. Inhibition of the renin-angiotensin system by amniotic fluid stem cells may contribute to these beneficial effects. In conclusion, treatment with amniotic fluid stem cells may be beneficial in kidney diseases characterized by progressive renal fibrosis.

Renal Extracellular Matrix Scaffolds From Discarded Kidneys Maintain Glomerular Morphometry and Vascular Resilience and Retains Critical Growth Factors.

Background Extracellular matrix (ECM) scaffolds, obtained through detergent-based decellularization of native kidneys, represent the most promising platform for investigations aiming at manufacturing kidneys for transplant purposes. We previously showed that decellularization of the human kidney yields renal ECM scaffolds (hrECMs) that maintain their basic molecular components, are cytocompatible, stimulate angiogenesis, and show an intact innate vasculature. However, evidence that the decellularization preserves glomerular morphometric characteristics, physiological parameters (pressures and resistances of the vasculature bed), and biological properties of the renal ECM, including retention of important growth factors (GFs), is still missing. Methods To address these issues, we studied the morphometry and resilience of hrECMs’ native vasculature with resin casting at electronic microscopy and pulse-wave measurements, respectively. Moreover, we determined the fate of 40 critical GFs post decellularization with a glass chip-based multiplex enzyme-linked immunosorbent assay array and in vitro immunofluorescence. Results Our method preserves the 3-dimensional conformation of the native glomerulus. Resin casting and pulse-wave measurements, showed that hrECMs preserves the microvascular morphology and morphometry, and physiological function. Moreover, GFs including vascular endothelial growth factor and its receptors are retained within the matrices. Conclusions Our results indicate that discarded human kidneys are a suitable source of renal scaffolds because they maintain a well-preserved structure and function of the vasculature, as well as GFs that are fundamental to achieve a satisfying recellularization of the scaffold in vivo due to their angiogenic properties.