YEAR IN REVIEW STEM CELLS IN 2013
Potential use of stem or progenitor cells for kidney regeneration Luigi Biancone and Giovanni Camussi
2013 saw the publication of numerous studies that identified resident renal stem or progenitor cells, induced pluripotent stem cells and strategies based on stem cell paracrine action, which all might be suitable for kidney regeneration after injury. Biancone, L. & Camussi, G. Nat. Rev. Nephrol. 10, 67–68 (2014); published online 3 December 2013; doi:10.1038/nrneph.2013.257
A new frontier of medicine is the regener ation of injured organs. Nephrologists dream of clinical strategies to repair dif ferent nephron compartments after acute and chronic injury. Preclinical studies have shown the beneficial effects of treat ment with stem cells of various origins. In the past decade, researchers have focused extensively on identifying resident stem or progenitor cells within the kidney to investigate their regenerative potential for use in stem cell-based therapies. Renal pro genitor cells have been identified in rodents and humans, but despite their ability to localize within the kidney after injury or during development, their role in regenera tion and their potential therapeutic appli cations are still elusive. However, most of these progenitor cells do not fulfil the cri teria that define true stem cells, such as self renewal, clonogenicity and multipotential differentiation capacity, but can instead be considered as precursors of differentiated renal epithelial cells. Research carried out in 2013 has furthered our understanding of the resident stem cell c ontribution to renal regeneration. One key study identified a novel c‑Kit+ (mast/stem cell growth factor receptor Kit) cell population, localized to the thick ascending limb of the loop of Henle in neonatal rat kidneys, which displayed the characteristic properties of stem cells and generated both mesoderm and ectoderm progeny. 1 Moreover, when administered in rats with acute ischaemia–reperfusion injury, in vitro expanded c‑Kit+ cells con tributed to kidney recovery, by exerting a paracrine action and engraftment within renal structures such as glomeruli, vessels and tubules (Figure 1). Whether a similar
stem cell population is present in human kidneys remains to be determined, but if so, it would have important biological and therapeutic implications. Stem cells are responsible for nephrogenesis in humans up to the 34th week of gestation. However, a cell population with the same proper ties of putative fetal stem cells has not yet been detected in the adult kidney, despite the identification of renal progenitor cells expressing CD133, CD24 and nestin in different sites of the nephron.2,3 Another study identified a highly clonogenic and self-renewing epithelial nephron progenitor population from mid- gestational human fetal kidneys, which defined a source of cells with potential for use in the regeneration of nephron epi thelial structures (loop of Henle, proximal
and distal tubules).4 After engraftment onto a chick embryo chorioallantoic membrane, these cells initia ted tubulogenesis and showed nephron epithelial differentiation potential. In vivo engraftment of these cells showed beneficial effects and improved renal function in the 5/6 chronic progres sive mouse renal injury model that mimics human chronic kidney disease. Notably, the regenera tive potential of human nephron progenitor cells in the remnant kidney was detected at just 3 months after their injection. Identification of renal stem or progeni tor cells in fetal or adult kidneys is relevant to understanding the regenerative poten tial of the kidney, but is limited as a thera peutic approach because the cell source is not easily accessible and only a potential Tubular specification
Resident stem cells derived from neonatal or fetal kidney Ex-vivo expanded neonatal rat c-Kit+ cells Human fetal kidney NCAM+ cells
hucMSC
Mesodermal development
iPSCs or GPSCs
Engraftment
Engraftment Differentiation Kidney regeneration
AKI recovery
De-differentiation Proliferation
Exosomes
Tubular cell injury
Re-differentiation
Activation of ERK1/2
Tubular epithelial cell repair
Figure 1 | Strategies for kidney regeneration. Abbreviations: AKI, acute kidney injury; c‑Kit, mast/stem cell growth factor receptor Kit; ERK1, mitogen-activated protein kinase 3; ERK2, mitogen-activated protein kinase 1; GPSC, germline cell-derived pluripotent stem cell; hucMSC, human umbilical cord mesenchymal stem cell; iPSC, induced pluripotent stem cell; NCAM, neural cell adhesion molecule 1.
NATURE REVIEWS | NEPHROLOGY
VOLUME 10 | FEBRUARY 2014 | 67 © 2014 Macmillan Publishers Limited. All rights reserved
YEAR IN REVIEW Key advances ■■ Stem or progenitor cells derived from neonatal or fetal kidneys have regenerative potential in acute and chronic models of kidney injury, acting either by paracrine mechanisms or by permanent engraftment1,4 ■■ New strategies have been developed to induce nephrogenic differentiation of induced pluripotent stem cells5,7 ■■ The use of extracellular vesicles derived from stem cells has been shown to be a potential therapeutic approach for acute kidney injury by stimulating endogenous kidney repair10
allogeneic therapy can be envisaged. Alternative sources of stem or progenitor cells, which bypass these limitations must therefore be identified. One such strategy is to develop human induced pluripotent stem cells (hiPSCs) with nephrogenic potential. An important study in this area of research achieved an efficient induc tion of nephrogenic intermediate meso derm generated f rom hiPSCs. 5 The researchers established a system of homo log ous recombin ation in hiPSCs using bacterial artificial chromosome vectors and a genomic DNA analysis array based on detection of single-nucleotide polymorph isms. This system generated green fluores cent protein reporter cell lines in hiPSCs for protein odd-skipped-related 1, an early marker of intermediate mesoderm. These cells were used for monitoring differen tiation of the cell lines. By using combi national treatments with growth factors, the investigators established a protocol for inducing intermediate mesoderm from hiPSCs with up to 90% of cells positive for protein odd-skipped-related 1, which is the first step that leads to cells of the renal lineage. This study demonstrated the feasi bility of monitoring the nephrogenic differ entiation capacity of hiPSCs and provides a new strategy for investigating the efficiency and specificity of methods to obtain renal differentiation of hiPSCs. Other studies have generated germ line cell-derived pluripotent stem cells (GPSCs) from both human and mouse adult spermatogonial stem cells. 6 These cells share characteristics with embryonic stem cells at both the cellular and molecular level and display high plasticity—they can differentiate into hepatocytes, haemato poietic cells, neurons, cardiomyocytes, smooth muscle cells and endothelial cells. In a pivotal study, researchers investigated 68 | FEBRUARY 2014 | VOLUME 10
whether GPSCs derived from adult spermatogonial stem cells could be used for renal regeneration.7 Using a novel renal epithelial cell differentiation protocol, the researchers obtained in vitro functional renal tubular-like cells from mice. Injection of GPSC-derived tubular-like cells into mice undergoing unilateral nephrectomy, followed by ischaemia–reperfusion injury in the remaining kidney, protected against acute and chronic renal damage. These results suggest that GPSC-derived tubularlike cells are functionally active in vivo, enabling the repair of renal damage in a murine model of ischaemia–reperfusion injury. Such techniques open the possibil ity of an autologous strategy for repairing damage in acute renal disease, with the added advantage of using GPSCs directly isolated from patients (limited to males). Other potential sources of autologous or heterologous stem cells, including mesen chymal stem cells derived from bone marrow, adipose tissue, umbilical cord vein and placenta, have been extensively investi gated in preclinical models and phase I clinical trials. Despite experimental data indicating a beneficial effect of these cells in kidney regeneration, the mechanisms involved remain controversial. Studies in transgenic mice models suggest epithelial cells that survive after acute kidney injury can aid tubular repair. 8 The paracrine theory has recently changed the interpreta tion of stem cell action and potential applications in regenerative medicine. Extracellular vesicles, including exosomes derived from the endosomal compartment and microvesicles released from the cell surface, have a key role in the interaction between stem and injured cells.9 The role of vesicles as vehicles of cell-to-cell com munication has been well established, with Nobel prizes awarded to Rothman, Schekman and Südhof in 2013 “for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells”. Vesicles that convey extrac ellular RNA might lead to transfer of genetic infor mation between cells.9 Preliminary studies have demonstrated vesicles derived from mesenchymal stem cells might mimic the effect of these cells and confer a stem celllike phenotype on injured cells, with the consequent activation of self-regenerative programmes involving cell cycle re-entry and differentiation. 9 Exosomes released from human umbilical cord mesenchymal stem cells limit cisplatin nephrotoxicity by decreasing oxidative stress-induced
apoptosis of renal cells, and promote proliferation of damaged tubular cells by activating the mitogen-activated protein kinase (ERK1/2) pathway. 10 This study suggests that exosomes could be exploited as a potential therapeutic tool in acute kidney injury. In conclusion, studies in 2013 have demonstrated the potential use of resident stem or progenitor cells and induced pluri potent stem cells for kidney regeneration. An additional alternative strategy involves stimulation of the intrinsic regenerative properties of the kidney by vesicle-induced reprogramming of injured cells. Department of Medical Sciences, University of Torino, Corso Dogliotti 14, 10126 Turin, Italy (L. Biancone, G. Camussi). Correspondence to: G. Camussi [email protected] Competing interests G. Camussi is a named inventor on patents US2011256111 (A1) and ES2423483 (T3). See the article online for full details of the patents. L. Biancone declares no competing interests. 1.
Rangel, E. B. et al. C-Kit+ cells isolated from developing kidneys are a novel population of stem cells with regenerative potential. Stem Cells 31, 1644–1656 (2013). 2. Bussolati, B. et al. Isolation of renal progenitor cells from adult human kidney. Am. J. Pathol. 166, 545–555 (2005). 3. Sagrinati, C. et al. Isolation and characterization of multipotent progenitor cells from the Bowman’s capsule of adult human kidneys. J. Am. Soc. Nephrol. 17, 2443–2456 (2006). 4. Harari-Steinberg, O. et al. Identification of human nephron progenitors capable of generation of kidney structures and functional repair of chronic renal disease. EMBO Mol. Med. 5, 1556–1568 (2013). 5. Mae, S. et al. Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells. Nat. Commun. http://dx.doi.org/10.1038/ncomms2378. 6. Fagoonee, S., Pellicano, R., Silengo, L. & Altruda, F. Potential applications of germline cell-derived pluripotent stem cells in organ regeneration. Organogenesis 7, 116–122 (2011). 7. De Chiara, L. et al. Renal cells from spermatogonial germline stem cells protect against kidney injury. J. Am. Soc. Nephrol. http://dx.doi.org/10.1681/ ASN.2013040367. 8. Humphreys, B. D. et al. Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell 2, 284–291 (2008). 9. Camussi, G. et al. Exosomes/microvesicles as a mechanism of cell‑to‑cell communication. Kidney Int. 78, 838–848 (2010). 10. Zhou, Y. et al. Exosomes released by human umbilical cord mesenchymal stem cells protect against cisplatin-induced renal oxidative stress and apoptosis in vivo and in vitro. Stem Cell Res. Ther. http://dx.doi.org/10.1186/ scrt194.
www.nature.com/nrneph © 2014 Macmillan Publishers Limited. All rights reserved
Potential use of stem or progenitor cells for kidney regeneration Luigi Biancone and Giovanni Camussi
2013 saw the publication of numerous studies that identified resident renal stem or progenitor cells, induced pluripotent stem cells and strategies based on stem cell paracrine action, which all might be suitable for kidney regeneration after injury. Biancone, L. & Camussi, G. Nat. Rev. Nephrol. 10, 67–68 (2014); published online 3 December 2013; doi:10.1038/nrneph.2013.257
A new frontier of medicine is the regener ation of injured organs. Nephrologists dream of clinical strategies to repair dif ferent nephron compartments after acute and chronic injury. Preclinical studies have shown the beneficial effects of treat ment with stem cells of various origins. In the past decade, researchers have focused extensively on identifying resident stem or progenitor cells within the kidney to investigate their regenerative potential for use in stem cell-based therapies. Renal pro genitor cells have been identified in rodents and humans, but despite their ability to localize within the kidney after injury or during development, their role in regenera tion and their potential therapeutic appli cations are still elusive. However, most of these progenitor cells do not fulfil the cri teria that define true stem cells, such as self renewal, clonogenicity and multipotential differentiation capacity, but can instead be considered as precursors of differentiated renal epithelial cells. Research carried out in 2013 has furthered our understanding of the resident stem cell c ontribution to renal regeneration. One key study identified a novel c‑Kit+ (mast/stem cell growth factor receptor Kit) cell population, localized to the thick ascending limb of the loop of Henle in neonatal rat kidneys, which displayed the characteristic properties of stem cells and generated both mesoderm and ectoderm progeny. 1 Moreover, when administered in rats with acute ischaemia–reperfusion injury, in vitro expanded c‑Kit+ cells con tributed to kidney recovery, by exerting a paracrine action and engraftment within renal structures such as glomeruli, vessels and tubules (Figure 1). Whether a similar
stem cell population is present in human kidneys remains to be determined, but if so, it would have important biological and therapeutic implications. Stem cells are responsible for nephrogenesis in humans up to the 34th week of gestation. However, a cell population with the same proper ties of putative fetal stem cells has not yet been detected in the adult kidney, despite the identification of renal progenitor cells expressing CD133, CD24 and nestin in different sites of the nephron.2,3 Another study identified a highly clonogenic and self-renewing epithelial nephron progenitor population from mid- gestational human fetal kidneys, which defined a source of cells with potential for use in the regeneration of nephron epi thelial structures (loop of Henle, proximal
and distal tubules).4 After engraftment onto a chick embryo chorioallantoic membrane, these cells initia ted tubulogenesis and showed nephron epithelial differentiation potential. In vivo engraftment of these cells showed beneficial effects and improved renal function in the 5/6 chronic progres sive mouse renal injury model that mimics human chronic kidney disease. Notably, the regenera tive potential of human nephron progenitor cells in the remnant kidney was detected at just 3 months after their injection. Identification of renal stem or progeni tor cells in fetal or adult kidneys is relevant to understanding the regenerative poten tial of the kidney, but is limited as a thera peutic approach because the cell source is not easily accessible and only a potential Tubular specification
Resident stem cells derived from neonatal or fetal kidney Ex-vivo expanded neonatal rat c-Kit+ cells Human fetal kidney NCAM+ cells
hucMSC
Mesodermal development
iPSCs or GPSCs
Engraftment
Engraftment Differentiation Kidney regeneration
AKI recovery
De-differentiation Proliferation
Exosomes
Tubular cell injury
Re-differentiation
Activation of ERK1/2
Tubular epithelial cell repair
Figure 1 | Strategies for kidney regeneration. Abbreviations: AKI, acute kidney injury; c‑Kit, mast/stem cell growth factor receptor Kit; ERK1, mitogen-activated protein kinase 3; ERK2, mitogen-activated protein kinase 1; GPSC, germline cell-derived pluripotent stem cell; hucMSC, human umbilical cord mesenchymal stem cell; iPSC, induced pluripotent stem cell; NCAM, neural cell adhesion molecule 1.
NATURE REVIEWS | NEPHROLOGY
VOLUME 10 | FEBRUARY 2014 | 67 © 2014 Macmillan Publishers Limited. All rights reserved
YEAR IN REVIEW Key advances ■■ Stem or progenitor cells derived from neonatal or fetal kidneys have regenerative potential in acute and chronic models of kidney injury, acting either by paracrine mechanisms or by permanent engraftment1,4 ■■ New strategies have been developed to induce nephrogenic differentiation of induced pluripotent stem cells5,7 ■■ The use of extracellular vesicles derived from stem cells has been shown to be a potential therapeutic approach for acute kidney injury by stimulating endogenous kidney repair10
allogeneic therapy can be envisaged. Alternative sources of stem or progenitor cells, which bypass these limitations must therefore be identified. One such strategy is to develop human induced pluripotent stem cells (hiPSCs) with nephrogenic potential. An important study in this area of research achieved an efficient induc tion of nephrogenic intermediate meso derm generated f rom hiPSCs. 5 The researchers established a system of homo log ous recombin ation in hiPSCs using bacterial artificial chromosome vectors and a genomic DNA analysis array based on detection of single-nucleotide polymorph isms. This system generated green fluores cent protein reporter cell lines in hiPSCs for protein odd-skipped-related 1, an early marker of intermediate mesoderm. These cells were used for monitoring differen tiation of the cell lines. By using combi national treatments with growth factors, the investigators established a protocol for inducing intermediate mesoderm from hiPSCs with up to 90% of cells positive for protein odd-skipped-related 1, which is the first step that leads to cells of the renal lineage. This study demonstrated the feasi bility of monitoring the nephrogenic differ entiation capacity of hiPSCs and provides a new strategy for investigating the efficiency and specificity of methods to obtain renal differentiation of hiPSCs. Other studies have generated germ line cell-derived pluripotent stem cells (GPSCs) from both human and mouse adult spermatogonial stem cells. 6 These cells share characteristics with embryonic stem cells at both the cellular and molecular level and display high plasticity—they can differentiate into hepatocytes, haemato poietic cells, neurons, cardiomyocytes, smooth muscle cells and endothelial cells. In a pivotal study, researchers investigated 68 | FEBRUARY 2014 | VOLUME 10
whether GPSCs derived from adult spermatogonial stem cells could be used for renal regeneration.7 Using a novel renal epithelial cell differentiation protocol, the researchers obtained in vitro functional renal tubular-like cells from mice. Injection of GPSC-derived tubular-like cells into mice undergoing unilateral nephrectomy, followed by ischaemia–reperfusion injury in the remaining kidney, protected against acute and chronic renal damage. These results suggest that GPSC-derived tubularlike cells are functionally active in vivo, enabling the repair of renal damage in a murine model of ischaemia–reperfusion injury. Such techniques open the possibil ity of an autologous strategy for repairing damage in acute renal disease, with the added advantage of using GPSCs directly isolated from patients (limited to males). Other potential sources of autologous or heterologous stem cells, including mesen chymal stem cells derived from bone marrow, adipose tissue, umbilical cord vein and placenta, have been extensively investi gated in preclinical models and phase I clinical trials. Despite experimental data indicating a beneficial effect of these cells in kidney regeneration, the mechanisms involved remain controversial. Studies in transgenic mice models suggest epithelial cells that survive after acute kidney injury can aid tubular repair. 8 The paracrine theory has recently changed the interpreta tion of stem cell action and potential applications in regenerative medicine. Extracellular vesicles, including exosomes derived from the endosomal compartment and microvesicles released from the cell surface, have a key role in the interaction between stem and injured cells.9 The role of vesicles as vehicles of cell-to-cell com munication has been well established, with Nobel prizes awarded to Rothman, Schekman and Südhof in 2013 “for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells”. Vesicles that convey extrac ellular RNA might lead to transfer of genetic infor mation between cells.9 Preliminary studies have demonstrated vesicles derived from mesenchymal stem cells might mimic the effect of these cells and confer a stem celllike phenotype on injured cells, with the consequent activation of self-regenerative programmes involving cell cycle re-entry and differentiation. 9 Exosomes released from human umbilical cord mesenchymal stem cells limit cisplatin nephrotoxicity by decreasing oxidative stress-induced
apoptosis of renal cells, and promote proliferation of damaged tubular cells by activating the mitogen-activated protein kinase (ERK1/2) pathway. 10 This study suggests that exosomes could be exploited as a potential therapeutic tool in acute kidney injury. In conclusion, studies in 2013 have demonstrated the potential use of resident stem or progenitor cells and induced pluri potent stem cells for kidney regeneration. An additional alternative strategy involves stimulation of the intrinsic regenerative properties of the kidney by vesicle-induced reprogramming of injured cells. Department of Medical Sciences, University of Torino, Corso Dogliotti 14, 10126 Turin, Italy (L. Biancone, G. Camussi). Correspondence to: G. Camussi [email protected] Competing interests G. Camussi is a named inventor on patents US2011256111 (A1) and ES2423483 (T3). See the article online for full details of the patents. L. Biancone declares no competing interests. 1.
Rangel, E. B. et al. C-Kit+ cells isolated from developing kidneys are a novel population of stem cells with regenerative potential. Stem Cells 31, 1644–1656 (2013). 2. Bussolati, B. et al. Isolation of renal progenitor cells from adult human kidney. Am. J. Pathol. 166, 545–555 (2005). 3. Sagrinati, C. et al. Isolation and characterization of multipotent progenitor cells from the Bowman’s capsule of adult human kidneys. J. Am. Soc. Nephrol. 17, 2443–2456 (2006). 4. Harari-Steinberg, O. et al. Identification of human nephron progenitors capable of generation of kidney structures and functional repair of chronic renal disease. EMBO Mol. Med. 5, 1556–1568 (2013). 5. Mae, S. et al. Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells. Nat. Commun. http://dx.doi.org/10.1038/ncomms2378. 6. Fagoonee, S., Pellicano, R., Silengo, L. & Altruda, F. Potential applications of germline cell-derived pluripotent stem cells in organ regeneration. Organogenesis 7, 116–122 (2011). 7. De Chiara, L. et al. Renal cells from spermatogonial germline stem cells protect against kidney injury. J. Am. Soc. Nephrol. http://dx.doi.org/10.1681/ ASN.2013040367. 8. Humphreys, B. D. et al. Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell 2, 284–291 (2008). 9. Camussi, G. et al. Exosomes/microvesicles as a mechanism of cell‑to‑cell communication. Kidney Int. 78, 838–848 (2010). 10. Zhou, Y. et al. Exosomes released by human umbilical cord mesenchymal stem cells protect against cisplatin-induced renal oxidative stress and apoptosis in vivo and in vitro. Stem Cell Res. Ther. http://dx.doi.org/10.1186/ scrt194.
www.nature.com/nrneph © 2014 Macmillan Publishers Limited. All rights reserved
