Stefano Da Sacco

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.

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.

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.

Stem cell and regenerative science applications in the development of bioengineering of renal tissue.

A rising number of patients with acute and chronic renal failure worldwide have created urgency for clinicians and investigators to search out alternative therapies other than chronic renal dialysis and/or organ transplantation. This review focuses on the recent achievements in this area, and discusses the various approaches in the development of bioengineering of renal tissue including recent discoveries in the field of regenerative medicine research and stem cells. A variety of stem cells, ranging from embryonic, bone marrow, endogenous, and amniotic fluid, have been investigated and may prove useful as novel alternatives for organ regeneration both in vitro and in vivo. Tissue engineering, developmental biology, and therapeutic cloning techniques have significantly contributed to our understanding of some of the molecular mechanisms involved in renal regeneration and have demonstrated that renal tissue can be generated de novo with similar physiologic functions as native tissue. Ultimately all of these emerging technologies may provide viable therapeutic options for regenerative medicine applications focused on the bioengineering of renal tissue for the future.

β-Cell Regeneration Mediated by Human Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) have been shown to ameliorate diabetes in animal models. The mechanism, however, remains largely unknown. An unanswered question is whether BMSCs are able to differentiate into β-cells in vivo, or whether BMSCs are able to mediate recovery and/or regeneration of endogenous β-cells. Here we examined these questions by testing the ability of hBMSCs genetically modified to transiently express vascular endothelial growth factor (VEGF) or pancreatic-duodenal homeobox 1 (PDX1) to reverse diabetes and whether these cells were differentiated into β-cells or mediated recovery through alternative mechanisms. Human BMSCs expressing VEGF and PDX1 reversed hyperglycemia in more than half of the diabetic mice and induced overall improved survival and weight maintenance in all mice. Recovery was sustained only in the mice treated with hBMSCs-VEGF. However, de novo β-cell differentiation from human cells was observed in mice in both cases, treated with either hBMSCs-VEGF or hBMSCs- PDX1, confirmed by detectable level of serum human insulin. Sustained reversion of diabetes mediated by hBMSCs-VEGF was secondary to endogenous β-cell regeneration and correlated with activation of the insulin/IGF receptor signaling pathway involved in maintaining β-cell mass and function. Our study demonstrated the possible benefit of hBMSCs for the treatment of insulin-dependent diabetes and gives new insight into the mechanism of β-cell recovery after injury mediated by hBMSC therapy.

The Milieu of Damaged Alveolar Epithelial Type 2 Cells Stimulates Alveolar Wound Repair by Endogenous and Exogenous Progenitors

Alveolar epithelial integrity is dependent upon the alveolar milieu, yet the milieu of the damaged alveolar epithelial cell type 2 (AEC2) has been little studied. Characterization of its components may offer the potential for ex vivo manipulation of stem cells to optimize their therapeutic potential. We examined the cytokine profile of AEC2 damage milieu, hypothesizing that it would promote endogenous epithelial repair while recruiting cells from other locations and instructing their engraftment and differentiation. Bronchoalveolar lavage and lung extract from hyperoxic rats represented AEC2 in vivo damage milieu, and medium from a scratch-damaged AEC2 monolayer represented in vitro damage. CINC-2 and ICAM, the major cytokines detected by proteomic cytokine array in AEC2 damage milieu, were chemoattractive to normoxic AECs and expedited in vitro wound healing, which was blocked by their respective neutralizing antibodies. The AEC2 damage milieu was also chemotactic for exogenous uncommitted human amniotic fluid stem cells (hAFSCs), increasing migration greater than 20-fold. hAFSCs attached within an in vitro AEC2 wound and expedited wound repair by contributing cytokines migration inhibitory factor and plasminogen activator inhibitor 1 to the AEC2 damage milieu, which promoted wound healing. The AEC2 damage milieu also promoted differentiation of a subpopulation of hAFSCs to express SPC, TTF-1, and ABCA3, phenotypic markers of distal alveolar epithelium. Thus, the microenvironment created by AEC2 damage not only promotes autocrine repair but also can attract uncommitted stem cells, which further augment healing through cytokine secretion and differentiation.

A Novel Source of Cultured Podocytes

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.

Amniotic fluid as a source of pluripotent and multipotent stem cells for organ regeneration

Purpose of reviewAmniotic fluid, due to its contact to the fetus during development, is considered an important diagnostic tool to evaluate the health status of the fetus during pregnancy. However, amniotic fluid also contains a heterogeneous cellular population that can be safely collected by amniocentesis and easily cultured. Many different cell types have been found within amniotic fluid and currently some of them are being tested for their possible use for cellular therapy. Recent findingsPotential of pluripotent and multipotent cells isolated from the amniotic fluid has been tested and in-vitro differentiations toward various cell types have been successfully performed. Furthermore, in-vivo studies are highlighting the benefits and mechanisms of amniotic fluid cells for therapy, with particular focus on kidney and lung diseases. SummaryAmniotic fluid may represent a precious source for easily and safely retrievable cell types that may be used for regenerative medicine purposes.

Regenerative medicine of the kidney

End stage renal disease is a major health problem in this country and worldwide. Although dialysis and kidney transplantation are currently used to treat this condition, kidney regeneration resulting in complete healing would be a desirable alternative. In this review we focus our attention on current therapeutic approaches used clinically to delay the onset of kidney failure. In addition we describe novel approaches, like Tissue Engineering, Stem cell Applications, Gene Therapy, and Renal Replacement Therapy that may one day be possible alternative therapies for patients with the hope of delaying kidney failure or even stopping the progression of renal disease.