Amniotic Fluid Stem Cells for Kidney Regeneration

Chapter 6

Amniotic Fluid Stem Cells for Kidney Regeneration Valentina Villani, Astgik Petrosyan, Roger E. De Filippo and Stefano Da Sacco GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics in Urology, Children’s Hospital Los Angeles, Division of Urology, Saban Research Institute, University of Southern California, Los Angeles, CA, United States

Chapter Outline Introduction Amniotic Fluid Amniotic Fluid Composition Amniotic Fluid Cells Amniotic Fluid Stem Cells Amniotic Fluid Mesenchymal Stem Cells Differentiation of Amniotic Fluid Stem Cells and Amniotic Fluid Mesenchymal Stem Cells Into Renal Lineages Amniotic Fluid Progenitors Amniotic Fluid Cells and Immunomodulatory Activity

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Amniotic Fluid Pluripotent Cells and Kidney Therapy Amniotic Fluid Stem Cells for the Treatment of Acute Kidney Injury Amniotic Fluid Stem Cells for the Treatment of Chronic Kidney Disease Amniotic Fluid Cell Applications in Kidney Bioengineering Conclusions List of Acronyms and Abbreviations References Further Reading

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INTRODUCTION The kidney presents a limited capacity to repair and recover following mild injury [1]. Following extensive or recurrent damage, the exogenous reparative mechanisms are insufficient to halt progression of renal injury. When loss of kidney functionality becomes preponderant, progression turns into an irreversible process, leading to kidney failure. Unfortunately, current therapies for the treatment of kidney failure are insufficient to halt disease progression, and ultimately, dialysis or transplantation is required. Cellular therapies have been explored as an alternative approach for the treatment of kidney damage with potential to slow down disease progression with promising results. In fact, different populations of pluripotent cells including bone marrowederived hematopoietic stem cells [2,3], mesenchymal stem cells (MSCs) [4,5], adipose-derived stem cells [6,7], endothelial progenitor cells [8e10], and amniotic fluid (AF)ederived stem cells [11e13] have been successfully tested in preclinical models of acute and chronic kidney injury. In particular, within AF is present a heterogeneous population that includes mesenchymal cells, organ-specific progenitor cells, and mature cells. AF cells present several advantages: they can be easily collected by amniocentesis, expanded, and characterized in culture with no ethical concerns [14]. Importantly, pluripotent stem cells and progenitors have been successfully differentiated into adult tissue types and have been extensively tested for the treatment of diseases affecting different organs, such as the lung, the pancreas, the kidney, and others. In this chapter, we will discuss the source, origin, differentiation potential, and regenerative and therapeutic role of AF cells to treat acute and chronic kidney diseases (CKDs). We will also report on the state of the art application of AF-derived cells with promising results for kidney regeneration and renal tissue engineering.

Perinatal Stem Cells. https://doi.org/10.1016/B978-0-12-812015-6.00006-6 Copyright © 2018 Elsevier Inc. All rights reserved.

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AMNIOTIC FLUID The AF is a clear, slightly yellowish liquid that fills the amniotic cavity and surrounds the unborn baby during pregnancy. The amnion membrane that encloses the developing fetus is composed by an outer layer of amniotic mesoderm and an inner layer of amniotic ectoderm [15,16]. The central role played by AF during fetal development has long been known: while providing nutrients and mechanical protection, acting like a cushion, it also prevents heat loss, acts as a barrier to infections, minimizes fetal scarring, and enables fetal movement and proper skeleton development [16]. Moreover, AF has also the fundamental role of supporting the development of several fetal organs, including the lung, the kidney, and the gut. The early embryo, while developing into a complex and multiorgan fetus, continuously interacts with AF, and as a response to fetal development, AF constantly changes its composition, solutes concentration, and volume. AF starts forming as early as 1 week postfertilization. It initially derives from maternal plasma, which passes through fetal membranes owing to hydrostatic and osmotic forces [16]. During embryogenesis, AF volume increases faster than embryonic size. The ratio between AF volume and fetal size is greater during the first weeks of gestation, and it progressively decreases in a linear fashion up to 28 weeks of gestation when the volume of the fluid plateaus at 800 mL and decreases to 400 mL at 42 weeks of gestation [16]. During the early stages of embryogenesis, as the placenta and fetal vessels develop, water and solutes pass from the mother through the placenta to the fetus and then to the AF. AF is therefore mainly generated by free bidirectional diffusion of fluids between the fetus, AF, placenta, and umbilical cord. AF composition is therefore similar to fetal plasma in the first weeks of development, as AF passes across the not-yet keratinized fetal skin. By 8 weeks of gestation, the fetus starts swallowing AF and the fetal kidneys begin to produce hypotonic urine, thus regulating AF volume. By 25 weeks of gestation, skin keratinization is complete and urine production is the main contributor to AF volume [16]. Importantly, when urine production by the fetus is inefficient because of kidney defects such as multicystic dysplastic kidney or bilateral renal agenesis, AF volume may drop to dangerously low levels, highlighting the importance of AF to sustain and promote fetal development. In fact, the outcome of pregnancies with early oligohydramnios is poor, which leads to either early rupture of the amnion membrane or early pregnancy termination [17]. Problems arise also by excessive accumulation of AF levels, a condition known as polyhydramnios, usually caused by infections, maternal diabetes, multiple pregnancies, or genetic diseases. Outcomes include preterm labor, early rupture of amnion membrane, or abnormal fetal presentation during labor [18]. However, other mechanisms most likely contribute to control AF volume during gestation, including intramembranous pathways that are responsible for the transfer of fluid and solutes from the AF to the fetus across the amniotic membranes. The human amnion is an epithelial layer that separates the amniotic cavity from the chorion. Initially flattened, the amniocytes that compose the amnion acquire a cuboidal cell shape and develop microvilli to increase their apical surface. Intercellular channels exist within the tight junctions of amniocytes and contribute to transmembranous diffusion. The existence of such mechanism explains the absence of polyhydramnios in conditions such as esophageal or duodenal atresia in the developing fetus, where AF diffusion is compensated by intramembranous diffusion.

AMNIOTIC FLUID COMPOSITION AF is mostly composed of water and other solutes such as electrolytes, proteins, carbohydrates, peptides, lipids, hormones, enzymes, and growth factors, which account for about 98% of its volume. The remaining 2% comprises a heterogeneous population of cells that includes MSCs, organ-specific progenitor cells, and mature differentiated cells. The cellular component within the AF derives from the developing fetus, specifically from tissues such as the lung and the gastrointestinal tract, which are consistently exposed to AF. Additionally, cells detaching from the forming kidney, excreted through the urine or exfoliating from the fetal skin, may contribute significantly to the AF cellular composition [14]. AF has been used as an important diagnostic tool since the 1970s when amniocentesis became available to assess fetal well-being. Amniocentesis is a minimally invasive prenatal procedure that can be used to determine the health of a developing fetus. It is performed by inserting an ultrasound-guided small needle into the amniotic sac to withdraw a small volume of AF (typically 15e20 mL of liquid) around the 15th to 20th week of gestation [16]. Following collection, the fluid is centrifuged and both the liquid and the cellular component can be analyzed and investigated to identify biomarkers of preterm labor or to determine the presence of infective processes. The most common evaluation of AF is performed to determine the presence of chromosomal abnormalities or neural tube defects and an array of inborn metabolic, hematologic, and genetic diseases. Along with its diagnostic use, AF has emerged in recent years as a novel source of stem and progenitor cells with therapeutic applicability. AF-derived cells present numerous advantages: safety of cell collection with no harm to the fetus; thus no ethical concern arises regarding their application, ease of cell expansion in vitro for several passages, differentiative

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capabilities, and importantly, the immunomodulatory properties that make these cells an ideal candidate to treat a wide array of diseases, spanning from chronic to acute injuries. As previously introduced, AF cells range from pluripotent stem cells to partially committed organ-specific progenitors, such as lung and renal progenitors, along with multiple mature cell types. The presence of cells from all three germ layers [19,20] as well as mesenchymal and hematopoietic progenitor cells [21] has been identified within the AF [14,22,23]. Interestingly, the AF cell pool has been shown to modify drastically during pregnancy, reflecting the organ and tissue changes undergoing during fetal growth. For example, cells expressing endodermal and mesodermal markers are predominantly present around 15 weeks of gestation and decrease in later weeks; on the other hand, cells expressing ectodermal markers have been found throughout the second trimester, possibly because of the contribution of the exfoliating fetal skin. Concurrent with the decrease of germ layer markers, expression of organspecific progenitor markers (such as TTF1, which is expressed in lung developmental cells; NKX2.5, expressed during heart development; or GDNF, expressed during nephrogenesis) has been found around 17e18 weeks of gestation. The expression of pluripotent markers (such as OCT4 and CD117) and mesenchymal markers (like CD90) remains constant, suggesting presence of stem and mesenchymal cells throughout the whole gestation period [14]. Thus, it is evident that AF harbors a diverse pool of cells that can be used, analyzed, and isolated at different times along the gestational time, offering an exciting tool for regenerative renal medicine purposes. In the following sections, we will discuss in detail the most widely used AF cell populations and focus on their uses in the renal regenerative field.

AMNIOTIC FLUID CELLS Because of the wide heterogenicity of cells residing within AF as well as the different isolation methods that can be used for the enrichment of pluripotent cells, several populations of stem cells have been identified within AF, with amniotic fluid stem cells (AFSCs) and amniotic fluid mesenchymal stem cells (AF-MSCs) being the most relevant in the regenerative medicine field.

Amniotic Fluid Stem Cells The most widely characterized AF-derived pluripotent stem cell population is identified by the expression of the stem cell marker c-kit (CD117). C-kit is the receptor for the stem cell factor and is expressed on the surface of a small fraction (1%) of the total amniotic cell pool. These cells, also known as AFSCs, were first identified and described in 2007 by De Coppi et al. [24]. After selection, either by FACS or by magnetic bead selection (MACS), about 1% of the total AF cell population is c-kitþ and could be further expanded in a-MEM supplemented with 20% Chang B and 2% Chang C solutions, 20% fetal bovine serum, 1% L-glutamine, and 1% antibiotics. AFSCs express markers characteristic of pluripotent cells with self-renewal capabilities such as octamer-binding transcription factor 4 (OCT4) and stage-specific embryonic antigen (SSEA4). Other surface markers that were found to be expressed by AFSCs include CD29, CD44, CD73, CD90, and CD105, which are typically expressed by mesenchymal and/or neural stem cells. Importantly, these cells were clonally derived by serial dilution and exhibited a high self-renewal capacity while lacking the ability to form teratomas when injected in vivo [24]. The potential of AFSCs to give rise in vitro to different lineages was demonstrated in numerous studies. AFSCs have been shown to differentiate into myocytes [24,25], cardiomyocytes [26], endothelial cells [27], osteoblasts [28e30], insulin-producing cells [31e34], or renal cells [35], although most of these protocols require multiple in vitro steps or genetic viral-mediated manipulation. More controversial is the capability of AFSCs to generate neural cells. Although the original publication confirmed acquisition of neuronal traits upon differentiation, other groups reported unsuccessful attempts. For example, in an attempt to further characterize c-kitþ cells, an extensive investigation on morphology changes and growth dynamics was performed by Arnhold et al. in comparing c-kit-selected cells with the negative fraction [36]. Although c-kitþ cells showed better differentiation toward adipocytes, osteocytes, and myocytes, neuronal differentiation was found more efficient in the negative population, suggesting that indeed the two fractions exhibit different characteristics [36]. Despite promising results in vitro confirming AFSCs’ plasticity as well as their putative potential to target a variety of diseases as functional substitutes, their application in vivo has risen questions on how exactly AFSCs function and exert their regenerative aid. The focus has shifted in most recent years on the ability of these cells to act mainly through paracrine mechanisms when applied in vivo. Immune tolerance coupled with paracrine properties makes AFSCs an ideal candidate, shifting the paradigm from cell therapy to paracrine action. Recently, our group evaluated the effect of decellularized kidney extracellular matrix (ECM) on differentiation and secretome profile of AFSCs and observed that the cellematrix interaction stimulates the secretion of several cytokines, chemokines, and soluble factors known for their renotropic activity such as PDGF-AA and VEGF [37] and IL6. However, the mechanism by which these growth factors could facilitate

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recovery and how stem cells can contribute to this process remains elusive. Additionally, recent investigations have demonstrated how AFSCs produce and secrete extracellular vesicles that contain a range of different molecules, including proteins, microRNAs, and other regulatory factors that contribute to modulate the surrounding milieu. Most of the studies that investigated the in vivo application of AFSCs as a therapeutic treatment to different injuries, including the kidney, the pancreas, and the lung, concur on the observation that a minimal number of injected cells effectively home to the target tissue and no cells are actually capable of full maturation [11,39,40]. The beneficial outcome observed by measure of physiological parameters is most likely the result of modulatory properties exerted by AFSCs.

Amniotic Fluid Mesenchymal Stem Cells Unlike AFSCs, AF-MSCs are not specifically selected for a stem cell marker. For this reason, a wide range of protocols have been established for the isolation and expansion of AF-MSCs. Different methodologies include, but are not limited to, the use of different culture media, growing surfaces, and derivation of single colony-forming clonal cell lines. On the other hand, versatility of both the source and the isolation has allowed the characterization of cells derived from several species including human [40e44], rodent [24,45,46], equine [47,48], ovine [49e51], bovine [52e54], caprine [55], canine [56,57], and porcine [58] sources. Although each of these sources has contributed greatly to our understanding of AF cells, for clarity, in this chapter we will mainly focus our attention on human- and rodent-derived AF-MSCs. One of the first methodologies for the isolation of AF-MSCs without the use of positive marker selections was established by Fauza’s group, which reported the isolation of unfractioned AF-MSCs from human samples between 20 and 37 weeks of gestation [59] Interestingly, AF-MSCs express similar markers as AFSCs and possess the same capacity to differentiate toward osteogenic, adipogenic, chondrogenic, hepatocyte-like, and neuronal cell linages. A growing number of studies have, since then, confirmed the specific markers expressed by AF-MSCs [41e44,60e63] using flow cytometry analysis. For example, a comprehensive study has shown that the large majority of AF-MSCs were positive for CD44, CD90, and CD147, partially positive for CD29, CD105, and CD106, and negative for CD34 and CD133 [64].

Differentiation of Amniotic Fluid Stem Cells and Amniotic Fluid Mesenchymal Stem Cells Into Renal Lineages The capacity of AFSCs and AF-MSCs to differentiate in vitro into different mature cells has been widely explored [24e35]. Investigators have also investigated the ability of AFSCs to differentiate into various renal lineages. The ability to undergo mesenchymal to epithelial transition after addition of EGF/PDGF and FGF4/HGF to the culture media was shown for AFSCs. Under these conditions, AFSCs express mesenchymal markers CD44 and CD29 concurrently with epithelial markers CD51 and ZO-1 and podocyte markers CD2AP and NPHS2 [65]. In another recent work, expression of renal markers SIX2, CD24, and KSP was confirmed in a stem cell population obtained by single colony-forming assay, confirming the presence of kidney-specific cells with pluripotent/multipotent traits [41]. However, replicating in vitro the full array of signals and interactions occurring within the developing kidney is challenging. To better recapitulate the specific niche of the developing renal compartment, investigators have tracked differentiation of AFSCs and AF-MSCs within developing kidneys with promising results. For example, we have previously shown that AFSCs can integrate within ex vivo developing murine kidney with long-term viability while contributing to the development of primordial renal structures such as renal vesicles and C- and S-shaped bodies. Moreover, 9 days postinjection, AFSCs were found to express early renal markers including ZO-1, GDNF, and claudin [35]. More recently, AFSCs, genetically modified to express GDNF with the use of adenovirus AxCAh-GDNF, were shown [64b] to differentiate toward a functional podocytelike cell expressing nestin, podocin, and a-actinin-4 and forming organized foot processes with intact interposed slit diaphragms in a model of chimeric aggregates of dissociated murine embryonic kidneys. GDNF-expressing AFSCs were further shown to be functional and internalize exogenously infused bovine serum albumin when implanted under the kidney capsule of uninephrectomized athymic rats.

Amniotic Fluid Progenitors Organ-specific developmental markers for the lung, the kidney, or the liver were found to be highly expressed in AF samples starting from 17 to 18 weeks of age [14]. Specifically, proteins such as WT-1 and ZO-1, GDNF, PAX-2, LIM-1, aquaporin-1, occludin, nephrin, and podocalyxin, which are expressed during kidney development, were found to be present in AF cell subpopulations, which raised the possibility of finding organ-derived progenitor cells that can be potentially isolated and applied for regenerative purposes to target specific tissues.

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Isolation and characterization of AF-derived renal progenitor cells is well-described FACS sorting of cells expressing both CD24 and OB-cadherin resulted in a population of cells with partially committed renal phenotype as demonstrated by the expression of markers such as ZO-1, GDNF, PAX-2, and LIM-1. Further selection within the CD24þ/OBcadherinþ cell pool for markers representative of mesangial, podocyte, and vascular progenitors confirmed the heterogeneity of such cell population and the presence of subpopulations of cells with different phenotypes, committed to different structures within the same organ/tissue [14]. In particular, we have previously described the isolation of cells expressing CD24, OB-cadherin, and podocalyxin, which possess a podocyte-committed phenotype and cells expressing SIX2 and CITED1 as nephron progenitors [65,66]. In vivo, podocytes are responsible for the production of collagen IV, the main component of the glomerular basement membrane, and, together with endothelial cells, form the glomerular filtration barrier. Several diseases lead to chronic inflammation and loss of podocytes, resulting in damaged filtration and proteinuria. The identification and characterization of human AF-derived podocyte precursors with the ability to differentiate into mature podocytes has opened the possibility to design and generate in vitro filtration units, mimicking the human glomerulus [65].

AMNIOTIC FLUID CELLS AND IMMUNOMODULATORY ACTIVITY Both AFSCs and AF-MSCs exhibit immunomodulatory properties comparable with MSCs. Moreover, AFSCs like other MSCs are found to be positive for class I major histocompatibility (MHC) antigens (HLA-ABC) and are negative for MHC class II (HLA-DR) or costimulatory molecules (CD80, CD86, CD40). Once activated, in vitro AFSCs express high levels of chemokines such as growth-related oncogene (GRO) family members and monocyte chemotactic protein-1 (MCP-1) to modulate inflammation and promote wound healing [67]. Like MSCs, AF-derived stem cells are capable of suppressing in vitro inflammatory responses by inhibiting lymphocyte activation [60,68]. Notably, AF-derived stem cells exert a stronger inhibition of T cell proliferation along with inhibition of Th1 polarization, counteracting disease progression and inflammation. In addition, AF-derived cells promote Th2 polarization with direct production of IL-10 and IL-4 while stimulating Th17 to increase production of IL-6 and IL-17, cytokines known for their beneficial effects on tissue regeneration and reduction of inflammatory response [60]. AFSCs seem to work in a dose-dependent manner and exhibit a stronger inhibition of T cell and NK cell proliferation and lower expression of HLA class I molecule and NK-activated ligands when obtained during the first trimester [60]. Importantly, an increasing number of studies are confirming the immunomodulatory potential of AF-derived cells and have opened new avenues for the clinical use of these cells as possible treatment for rejection in organ transplantation. In fact, despite recent advancements on short-term outcomes following renal transplantation, complications associated with long-term graft function caused by ischemia/reperfusion (I/ R) injury and antibody-mediated rejection remain a major challenge [69,70]. The possibility that stem cells, including AFderived cells, could prevent graft rejection by providing immune tolerance and reducing toxicity and I/R-associated injury in organ transplantation has been gaining interest worldwide, and their use has been tested in preliminary studies on animal models. In renal transplantation studies, AF-MSCs have been confirmed capable of inducing immune tolerance by inhibiting the proliferation of CD4þ and CD8þ cells and secretion of inflammatory factors IL-4 and IFN-g, preventing the infiltration of inflammatory cells and lowering oxidative stress level in rats [71]. Although studies in small animals can provide some useful insights on the potential of AF-derived cells for the prevention and treatment of graft rejection, preclinical studies in larger animals would provide additional support to their therapeutic capacity [72]. Unfortunately, stem cell therapy studies performed in large animals are scarce, and this is particularly true for AF cells. Administration of AF-MSCs in porcine model of kidney transplantation has been reported. In this work, injection of AF-MSCs 6 days after autologous transplantation provides strong protection against fibrosis and improves kidney function [13]. A significant increase in VEGF-A and angiotensin II, and an equivalent decrease in Flt1 expression in kidneys as late as 3 months after stem cell transplantation, suggests that the mechanism of action lies on the modulation of the proangiogenic pathway. This hypothesis was further confirmed in vitro, where AF-MSCs provided protection to I/R-injured endothelial cells by promoting survival and angiogenesis [13]. A more in-depth analysis of in vivo AF mechanisms of action in acute kidney disease (AKI) and CKD, including immunomodulation, is discussed in the following sections.

AMNIOTIC FLUID PLURIPOTENT CELLS AND KIDNEY THERAPY The increasing prevalence of end-stage renal failure and the lack of significant progress in therapeutic approaches during the last decades have prompted clinicians and scientists to find new therapies aimed at treating kidney diseases. AF has emerged as an alternative source of cells that have been shown to possess potential not only as a model to study renal differentiation but also as a tool to understand disease progression. The use of AF cells aimed at slowing down, halting, or

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reversing renal disease using in vivo animal models of acute and chronic kidney injury has been essential in our understanding of their therapeutic potential. The most relevant studies that have contributed to build our knowledge on their ability to migrate to the site of injury, exert immunomodulatory activity, and promote tissue regeneration are summarized in the following sections.

Amniotic Fluid Stem Cells for the Treatment of Acute Kidney Injury AKI is defined as the rapid and sudden decline in renal function occurring within few hours or days. Causes of AKI include infection of bacterial or viral origin, drug nephrotoxicity, physical kidney damage, sepsis, significant blood loss, or blockage of urinary tract and can lead to a rapid loss of renal function, decrease in renal filtration, organ failure and, in the long term, permanent renal damage [73]. Unfortunately, no specific pharmacologic therapy is effective in patients with established acute kidney injury, and therapies are limited to supportive treatment, including renal replacement therapy. However, despite the use of various pharmacologic agents, mortality rate of patients affected by AKI ranges between 25% and 70%, with peaks as high as 80% in patients requiring dialysis, [74]. Moreover, in patients undergoing renal replacement therapy, risk of hypotension is common and about 10% of the patients cannot be treated because of hemodynamic instability [75e79]. For this reason, alternative approaches for the treatment of AKI are urgently needed. Because one of the principal symptoms of AKI is a strong and rapid immune response, treatment with stem cells has been proposed for AKI prevention and early treatment. AF-derived cells have shown promising results in the treatment of AKI. Our group reported the beneficial effects of AFSCs (of human origin) in an experimental model of AKI [73]. Using a glycerol-induced rhabdomyolysis model of acute tubular necrosis (ATN) in immunodeficient (nu/nu) mice, we showed that simultaneous injection of both glycerol- and AFSC-ameliorated kidney injury. In particular, injection of AFSCs prevented peaks in creatinine or blood urea nitrogen (BUN) and protected tubular epithelial cells against cast formation and apoptosis while increasing their proliferation and regeneration. Cytokine analysis suggested that AFSCs exerted their effect by inhibiting proinflammatory cytokines and promoting increased expression of antiinflammatory interleukins, such as IL10, and IL1RA. Interestingly, AFSCs were found to express aquaporin-1 (proximal tubular marker) and GDNF (nephrogenic marker) within tubular and glomerular compartments, respectively, which might suggest differentiation of AFSCs into kidney-specific phenotype. However, it is unclear how expression of renal markers plays a role, if any, on their beneficial effect. Moreover, when AFSCs were injected during the acute phase, they were unable to rescue kidney damage as confirmed by measured levels of serum creatinine and BUN levels. In a similar study, using the same rhabdomyolysisinduced ATN model of AKI in SCID Balb/c mice, Hauser et al. [88] showed that intravenous injection of AF-MSCs 72 h after glycerol injection was able to induce tubular recovery by enhancing proliferation and reducing tubular endothelial cell apoptosis [61]. Results delivered by AF-MSCs were comparable with the beneficial effects offered by bone marrow MSCs; interestingly, AF-MSCs appeared to remain within the renal parenchyma for a longer amount of time while providing a more prolonged antiapoptotic effect than MSCs. In contrast to our results, however, AF-MSCs and MSCs did not integrate into renal structures but were only found within the renal interstitium [61]. Notably, although the two studies differ in their methodology, including derivation of cell lines and time of injection, they delivered comparable protection mediated by paracrine signals. Using a different injury system, the efficacy of AF-MSC-derived cells has been explored in a cisplatin-induced acute nephrotoxicity model in rats showing that a single cell injection is capable of accelerating kidney damage recovery, significantly improving renal function and reducing oxidative stress, thus enhancing the endogenous regenerative capacity of the kidney [80,81]. Interestingly, GDNF-preconditioned AFSCs displayed greater protection from cisplatin-induced nephrotoxicity in immunodeficient NOD-SCID mice [12]. As reported, pretreatment of cells with GDNF significantly improved renal function as well as tubular cell proliferation, performing better than unstimulated cells alone, possibly by activating stem cell cytoprotective and antioxidant pathways. In the same work, other local paracrine factors including IL-6, VEGF, SDF-1, and IGF-1 were found to be potentially involved in mediating beneficial effects of AFSCs [12]. Another relevant model of AKI is generated by performing I/R. Unfortunately, I/R is an inevitable occurrence, particularly, during kidney transplantation [82] and represents one of the major causes of acute renal failure in allografts and native kidneys equally [83]. Several studies have confirmed beneficial effects of AFSCs in I/R injury. Recently, Monteiro Carvalho Mori da Cunha et al. have shown that injection of clonal AF-MSC-positive for CD24, KSP, SIX2, and PAX2 exhibits strong protection against ischemic injury in 12-week-old male Wistar rats [41]. Intravenous injection of cells 6 h post-I/R injury exerted beneficial effects as early as 24 h after treatment as confirmed by a decrease in serum creatinine level as well as lower hyaline cast formation and tubular necrosis. Additionally, injection of AF-MSCs increased proliferation of tubular epithelial cells along with a decreased infiltration of macrophages and activation of myofibroblasts. After 2 months, treated animals presented lower level of microalbuminuria and fibrotic index compared with the untreated

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cohort [40]. Moreover, in a recent study, Monteiro Carvalho Mori da Cunha et al. demonstrated that VEGF upregulation in AF-MSCs could enhance protection from I/R injury by reducing inflammatory response as well as promoting angiogenic and promitotic mechanisms [84]. However, higher doses of VEGF-AF-MSC exhibited a deleterious effect on injury progression, suggesting that the beneficial response is dose dependent [84].

Amniotic Fluid Stem Cells for the Treatment of Chronic Kidney Disease CKD is characterized by a slow, often unstoppable, progression of kidney deterioration characterized by gradual development of interstitial fibrosis and glomerulosclerosis. Unfortunately, no definitive therapy is available for CKD, and although antihypertensive medications have been shown to slow down its progression, dialysis or transplantation is eventually required. Nonetheless, the high cost and frequent side effects of dialysis along with a chronic shortage of organ donors are major limitations to these treatments. Despite the growing interest in AF-derived stem cell therapies to reverse or slow down CKD, very little is known about their therapeutic potential. In fact, in the last decade, only a few studies have explored the use of AF cells in advanced fibrotic kidney diseases. In 2012 we reported for the first time that a single systemic injection of clonal AFSCs into mice with Alport syndrome before the onset of proteinuria was able to significantly slow down disease progression as confirmed by lower glomerular and interstitial fibrosis, improved kidney function, and prolonged survival in mice [11]. Our study also found a higher number of preserved podocytes in treated animals as well as increasing M2 (prohealing) polarization in macrophages. Interestingly, no de novo production of Col4a5(IV) collagen was detected with injection of AFSCs, suggesting that AFSCs might work mainly through endocrine/paracrine mechanisms to delay disease progression [11]. Investigating a different model of CKD, Sun et al. reported that administration of AF-MSCs into nu/nu mice with unilateral ureteral obstruction alleviated the progression of fibrosis within the renal tissue by increasing VEGF-mediated angiogenesis while decreasing HIF-1a and TGF-b1 expression [85]. Additionally, AF-MSCs improved proximal tubular cell density by promoting their proliferation and decreasing apoptosis. Taken together, these studies suggest that AFSCs are not integrating within the kidney tissue during CKD but rather act as transient regulators of the injury processes, promoting the activation of regenerative mechanisms through secretion of renotropic healing molecules.

AMNIOTIC FLUID CELL APPLICATIONS IN KIDNEY BIOENGINEERING The shortage of supply for transplantable organs, including kidneys, is a global health concern. In fact, despite important advances in transplantation, an average of 14 people die each day waiting for a kidney transplant [86]. The search for alternative approaches has led scientists and clinicians to explore the potential of pluripotent cells for the ex vivo and in vitro reconstruction of whole organs. AF progenitor cells’ and AFSCs’ plasticity toward differentiation into various cell types and their ability to modulate the immune response in vivo have made them a preferred choice in de novo bioengineering of kidneys. Behavior of AFSCs has been studied on renal ECM scaffolds obtained from decellularized human kidneys. Decellularization has been performed using a perfusion of detergents (sodium dodecyl sulfate, SDS) and enzymes (DNase) to completely remove resident cells while preserving the renal ECM architecture and composition [87]. Under these conditions, the architectural integrity and the composition of ECM, along with maintenance of glomerular morphometry, vascular resilience, and important growth factors, were confirmed. Interestingly, we found that the decellularized ECM scaffolds entrap growth factors essential for angiogenesis, immunomodulation, cell motility, differentiation, and proliferation such as VEGF, FGF, PDGF, GM-CSF, IGF, and TGFb [37]. Following static seeding of AFSCs onto these decellularized renal ECMs without additional renal differentiation components, we have shown that cells after 3 weeks appear to adhere, differentiate, proliferate, and be metabolically active. Notably, morphology of AFSCs was found to be related to their location within the renal ECM [37]. Moreover, after just 4 days of seeding, we detected in the media various chemokines and matrix remodeling proteins such as VEGF, IL-8, MMP2, and TIMP2, suggesting a secretory activity of AFSCs [37]. Importantly, the panel of cytokines expressed by the cultured AFSCs (that include GRO, VEGF, IL-8, TGFa, and PDGF) are recognized to be important modulators of inflammation, angiogenesis, and wound healing during disease progression in vivo.

CONCLUSIONS AF is an advantageous source that harbors a heterogeneous population of cells with potential for both in vitro and in vivo studies (Scheme 6.1). In vitro differentiation of amniotic pluripotent and multipotent cells toward renal lineages can be performed without genetic manipulation or long stepwise differentiation protocols, allowing investigations of renal cell

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SCHEME 6.1 Use of amniotic fluid (AF)ederived cells for renal regenerative medicine.

specification and differentiation. At the same time, in vivo applications of AF cells for treatment of various diseases, including kidney damage, have been instrumental not only for evaluating them as therapeutic tools but also for investigating mechanisms of renal repair after injury. However, despite the abundance of available data, several issues still hamper the translation of AF cells to clinical trials. In fact, the absence of a coherent and consistent nomenclature, the lack of standards for their isolation and expansion, and the use of cells derived from different species in various animal models render interpretation and comparison of the results particularly challenging. Although it has been widely established that the principal mechanism of renal protection in vivo is primarily characterized by a paracrine activity as opposed to integration and differentiation, the underlying mechanisms by which AF cells exert their beneficial effect are still elusive and require further investigation. Answering these fundamental questions will eventually define therapeutic potential and limits of AF-derived stem cells for clinical use and will hopefully provide the tools needed to counteract the global health threat posed by kidney diseases.

LIST OF ACRONYMS AND ABBREVIATIONS AF Amniotic fluid AF-MSCs Amniotic fluid mesenchymal stem cells AFSCs Amniotic fluid stem cells AKI Acute kidney failure ATN Acute tubular necrosis BUN Blood urea nitrogen CKD Chronic kidney disease ECM Extracellular matrix I/R Ischemia/reperfusion MSCs Mesenchymal stem cells

REFERENCES [1] Chang-Panesso M, Humphreys BD. Cellular plasticity in kidney injury and repair. Nat Rev Nephrol 2017;13(1):39e46. [2] Li L, Black R, Ma Z, Yang Q, Wang A, Lin F. Use of mouse hematopoietic stem and progenitor cells to treat acute kidney injury. Am J Physiol Renal Physiol 2012;302(1):F9e19. [3] Lin F, Cordes K, Li L, Hood L, Couser WG, Shankland SJ, et al. Hematopoietic stem cells contribute to the regeneration of renal tubules after renal ischemia-reperfusion injury in mice. J Am Soc Nephrol 2003;14(5):1188e99. [4] Semedo P, Correa-Costa M, Antonio Cenedeze M, Maria Avancini Costa Malheiros D, Antonia dos Reis M, Shimizu MH, et al. Mesenchymal stem cells attenuate renal fibrosis through immune modulation and remodeling properties in a rat remnant kidney model. Stem Cells 2009;27(12):3063e73.

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[5] Lee SR, Lee SH, Moon JY, Park JY, Lee D, Lim SJ, et al. Repeated administration of bone marrow-derived mesenchymal stem cells improved the protective effects on a remnant kidney model. Ren Fail 2010;32(7):840e8. [6] Sheashaa H, Lotfy A, Elhusseini F, Aziz AA, Baiomy A, Awad S, et al. Protective effect of adipose-derived mesenchymal stem cells against acute kidney injury induced by ischemia-reperfusion in Sprague-Dawley rats. Exp Ther Med 2016;11(5):1573e80. [7] Liu T, Zhang Y, Shen Z, Zou X, Chen X, Chen L, et al. Immunomodulatory effects of OX40Ig gene-modified adipose tissue-derived mesenchymal stem cells on rat kidney transplantation. Int J Mol Med 2017;39(1):144e52. [8] Chen B, Bo CJ, Jia RP, Liu H, Wu R, Wu J, et al. The renoprotective effect of bone marrow-derived endothelial progenitor cell transplantation on acute ischemia-reperfusion injury in rats. Transplant Proc 2013;45(5):2034e9. [9] Liang CJ, Shen WC, Chang FB, Wu VC, Wang SH, Young GH, et al. Endothelial progenitor cells derived from Wharton’s jelly of human umbilical cord attenuate ischemic acute kidney injury by increasing vascularization and decreasing apoptosis, inflammation, and fibrosis. Cell Transplant 2015;24(7):1363e77. [10] Sangidorj O, Yang SH, Jang HR, Lee JP, Cha RH, Kim SM, et al. Bone marrow-derived endothelial progenitor cells confer renal protection in a murine chronic renal failure model. Am J Physiol Renal Physiol 2010;299(2):F325e35. [11] Sedrakyan S, Da Sacco S, Milanesi A, Shiri L, Petrosyan A, Varimezova R, et al. Injection of amniotic fluid stem cells delays progression of renal fibrosis. J Am Soc Nephrol 2012;23(4):661e73. [12] Rota C, Imberti B, Pozzobon M, Piccoli M, De Coppi P, Atala A, et al. Human amniotic fluid stem cell preconditioning improves their regenerative potential. Stem Cells Dev 2012;21(11):1911e23. [13] Baulier E, Favreau F, Le Corf A, Jayle C, Schneider F, Goujon JM, et al. 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Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25(1):100e6. [25] Ghionzoli M, Repele A, Sartiani L, Costanzi G, Parenti A, Spinelli V, et al. Human amniotic fluid stem cell differentiation along smooth muscle lineage. FASEB J 2013;27(12):4853e65. [26] Velasquez Mao AJ, Tsao CJM, Monroe MN, Legras X, Bissig-Choisat B, Bissig KD, et al. Differentiation of spontaneously contracting cardiomyocytes from non-virally reprogrammed human amniotic fluid stem cells. PLoS One 2017;12(5):e0177824. [27] Tancharoen W, Aungsuchawan S, Pothacharoen P, Markmee R, Narakornsak S, Kieodee J, et al. Differentiation of mesenchymal stem cells from human amniotic fluid to vascular endothelial cells. Acta Histochem 2017;119(2):113e21. [28] De Rosa A, Tirino V, Paino F, Tartaglione A, Mitsiadis T, Feki A, et al. Amniotic fluid-derived mesenchymal stem cells lead to bone differentiation when cocultured with dental pulp stem cells. Tissue Eng A 2011;17(5e6):645e53. 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In vitro differentiation into insulin-producing ß-cells of stem cells isolated from human amniotic fluid and dental pulp. Dig Liver Dis 2013;45(8):669e76. [34] Mu XP, Ren LQ, Yan HW, Zhang XM, Xu TM, Wei AH, et al. Enhanced differentiation of human amniotic fluid-derived stem cells into insulinproducing cells in vitro. J Diabetes Investig 2017;8(1):34e43.

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[35] Perin L, Giuliani S, Jin D, Sedrakyan S, Carraro G, Habibian R, et al. Renal differentiation of amniotic fluid stem cells. Cell Prolif 2007;40(6):936e48. [36] Arnhold S, Glüer S, Hartmann K, Raabe O, Addicks K, Wenisch S, et al. Amniotic-fluid stem cells: growth dynamics and differentiation potential after a CD-117-based selection procedure. Stem Cells Int 2011;2011:715341. [37] Petrosyan A, Orlando G, Peloso A, Wang Z, Farney AC, Rogers G, et al. Understanding the bioactivity of stem cells seeded on extracellular matrix scaffolds produced from discarded human kidneys: a critical step towards a new generation bio-artificial kidney. CellR4 2015;3(1):e1401. [38] Deleted in review. [39] Homsi E, Ribeiro-Alves MA, Lopes de Faria JB, Dias EP. Interleukin-6 stimulates tubular regeneration in rats with glycerol-induced acute renal failure. Nephron 2002;92(1):192e9. [40] Villani V, Milanesi A, Sedrakyan S, Da Sacco S, Angelow S, Conconi MT, et al. Amniotic fluid stem cells prevent ß-cell injury. Cytotherapy 2014;16(1):41e55. [41] Monteiro Carvalho Mori da Cunha MG, Zia S, Oliveira Arcolino F, Carlon MS, Beckmann DV, Pippi NL, et al. Amniotic fluid derived stem cells with a renal progenitor phenotype inhibit interstitial fibrosis in renal ischemia and reperfusion injury in rats. PLoS One 2015;10(8):e0136145. [42] Gucciardo L, Ochsenbein-Kolble N, Ozog Y, Verbist G, Van Duppen V, Fryns JP, et al. A comparative study on culture conditions and routine expansion of amniotic fluid-derived mesenchymal progenitor cells. Fetal Diagn Ther 2013;34(4):225e35. [43] Montemurro T, Bossolasco P, Cova L, Zangrossi S, Calzarossa C, Buiatiotis S, et al. Molecular and phenotypical characterization of human amniotic fluid cells and their differentiation potential. Bio Med Mater Eng 2008;18(4e5):183e5. [44] Pipino C, Pierdomenico L, Di Tomo P, Di Giuseppe F, Cianci E, D’Alimonte I, et al. 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FURTHER READING [1] Organ Procurement and Transplantation Network e http://optn.transplant.hrsa.gov/.