Unformatted text preview: Amnio&c Fluid Stem Cells (AFSC) and Kidney Disease Department of Urology Saban Research Ins=tute University of Southern California Childrens Hospital Los Angeles Chronic Kidney Disease
26 million of Americans have CKD People with kidney failure undergo either 1.Drugs (such as ACE or cyclosporin) in long run result to be nephrotoxic 1.Dialysis, an arDficial blood-cleaning process (expensive, hours of treatment, three Dmes per week) 1.Transplanta=on to receive a healthy kidney from a donor (shortage of donors, lifelong immunosuppression) Alterna&ve approach: stem cells ? Amnio&c Fluid Stem Cells (AFSC)
Amniocentesis samples: no risk for the fetus and the mother, large amount of cells, easy to culture, they are pluripotent in vitro as well as in vivo, hAFSC present low immunogenic activity and do not form teratoma Characterization of the AFSC (Nature Biotch., 2007) In vitro Kidney differentiation of AFSC (Cell Proliferation, 2007) In vivo Kidney Regeneration (PlosOne, 2010) Progenitors from Amniotic Fluid (Journal of Urology, 2010) AFSC for lung Regeneration (Stem Cells, 2008) Renal regenera=on using 1. AFSC 2. Using AFKP Tissue Engineering In vivo In vitro Embryonic kidney model Chronic kidney disease Acute damage Glycerol model of acute tubular Transgenic mouse necrosis models of Alport syndrome ACUTE KIDNEY INJURY: ATN Glycerol-Mediated Acute Tubular Necrosis Glycerol (7-8ml/kg) induces Rhabdomyolysis (crush syndrome, muscular trauma, dehydration, sepsis, drug nephrotoxicity): release in the bloodstream myoglobin that is filtered by the glomeruli, this cause damage to the tubular and renal dysfunction. Dehydra=on IntesDnal Hyperperfusion Endotoxemia InflammaDon Renal vasoconstricDon Glomerular filteraDon pressure Intramuscular Enhancement of tubular water reabsorpDon
Tubular cast formaDon Uric acid Aciduria Tubular ischemia Fiber necrosis Free radical producDon Myoglobin glycerol injec=on Iron release Muscle destrucDon Perin et al, 2010 Proximal Tubule: S1 and S2 (renal cortex) ,S3 (renal medulla) (brush border, small lumen, more cytoplasm Distal Tubule: (no brush border, big lumen, less cytoplasm) Change in the physiological parameters of the kidney in the acute phase , such us BUN and creatinine Disruptions of the brush border (cast formation) In vivo luciferase detection of hAFSC in ATN damaged kidneys. Protective effect of injected hAFSC as determined by measurements of blood creatinine and BUN levels. Protective effect of injected hAFSC, determined by maintenance of the morphological structure in glycerol induced ATN damaged kidneys. Protective effect of injected hAFSC as determined by increase of proliferation and decrease of apoptotic tubular cells in glycerol induced ATN kidneys. Kidney tubular epithelium is restored without replacement with bone marrowderived cells during repair after ischemic injury (Bonventre J. 2005) Pathophysiology of Acute Kidney Injury: Roles of Potential Inhibitors of Inflammation (Bonventre J. 2007) Immunomodulatory effects of hAFSC once injected into glycerol induced ATN-damaged kidneys. Injected mice present Luciferase-labeled AFSC can be tracked in vivo Prevention of the acute phase Preservation of the morphology, creatinine and BUN level Decrease of apoptosis of tubular epithelial cells Increase of proliferation of tubular epithelial cells Increase of IL10, IL1RA, IL6 Decrease of IL12p70, IL16, IL27, IL23 Decrease of SDF-1, KC, M-CSF, JE, MIP-2 and MIP-5 The mechanism of renal protection is not reached by integration of injected cells but by immuno-modulation CHRONIC KIDNEY DISEASE Alport Syndrome: a model of chronic disease Alport syndrome derives from a mutaDon of the 3, 4 or 5 chains of type IV collagen, that consDtute basement membrane in the kidney, ear and eye Animal models, transgenic mice (B6.Cg-ColIV5-/- and 129-ColIV3-/-) that develop syndromes similar to human paDents and two canine models. There are genetically distinct chains (1-6) of ColIV and they assembly in trimers
GBM and ColIV1 2 2 (Embryonic, BM - except in rodents in neuromuscular junction) ColIV345 (GBM, Capsule of Bowman) ColIV5 26 (Distal Tubules and Collecting Duct, Capsule of Bowman) In Alport patients and in the animal models the ColIV345 is substitute with the ColIV1 2 2 in the GBM and CB Chronic Kidney disease: Alport Syndrome
B6.Cg-Col4a5tm1Yseg/J Therapeu=c approach one single systemic injec=on of ~ 1x106 before the onset of proteinuria 1. Life span? 2. Physiological and morphologycal renal parameters? 3. Which is the in vivo mechanism of acDon of AFSC? Life span and physiological parameters of injected mice Trichrome-Masson staining (EMC deposi=on) 22 Injected AS mice present: increased life-span amelioration of kidney physiological levels less production of collagen I decrease production of ECM decrease of cytokines and chemokines levels Take home message 1. AFSC is a new source of stem cell that posses the "standard" proprie=es that iden=fy them as a mesenchymal stem cell popula=on 2. They are easily isolated with no harm to the fetus and the mother 3. 4. They can be cultured and expanded for many popula=ons in culture maintain the expression of stem cell markers undifferen=ated state pluricapability to give rise to different mature cell types maintain the cell cycle and telomeric length can par=cipate to the development of different =ssues They are used in Regenera=ve Medicine Applica=on: kidney Lung Pancreas Heart Bone, etc. Lung regeneraDon and Dssue engineering Lung Development
A. B. C. D. E.
The primiDve lung anlage emerging as the laryngo-tracheal groove from the ventral surface of the primiDve foregut. The primiDve trachea separaDng dorso-ventrally from the primiDve esophagus as the two primary bronchial branches arise from the lateral aspects of the laryngo-tracheal groove. The embryonic larynx and trachea with the two primary bronchial branches separated dorso-ventrally from the embryonic esophagus. The primiDve lobar bronchi branching from the primary bronchi. A schemaDc rendering of the term fetal airway in human. Warburton et al., 2010 Lung Anatomy Epithelium: Airways Alveoli Endothelium: Vasculature IntersDDum: Mesenchyme Netter, F.H. Respiratory System. Volume 7, CIBA Collection of Medical Illustrations. 1979. 40 phenotypically disDnct cell lineages idenDfied in ConsideraDons for RegeneraDve Anatomy and physiology of regeneraDve target: structure & funcDon Target Niches: endogenous stem cells? Type of Injury or Disease Challenges Endogenous vs. Exogenous therapy Delivery strategies FuncDonal Regions of the Lung Dictate Conduc=ng Airways (0-16) Designed to withstand turbulent flow FuncDons: Warm and humidify inspired air Distribute inspired air to respiratory bronchioles Remove foreign material from inspired air Respiratory Bronchioles (17-23) DistribuDon of air (laminar flow) FuncDon: Gas exchange Stem Cell Niches within the Lung:
glands (SMG) 2. Tracheal basal cells 3./4. Variant Clara cells (Clarav)
1. Neuroendocrine bodies (NEB) 2. Bronchoalveolar duct juncDon (BADJ) 1. Airway submucosal 5. Alveolar epithelial type II cells (AECII) Lung Injury and Disease Type of Injury/Disease Dictates the PotenDal What type of injury/ disease has occurred? When did injury/disease occur? How far has injury/disease progressed? Is there enough funcDonal Dssue remaining for repair? Challenges in Lung RegeneraDon Complex structure Magnitude of different cell types Various disease states poorly understood Endogenous vs. Exogenous Cellular Delivery Strategies Belur et al., Nature Protocols 2, 3146 - 3152 (2007) Penn-Century TM (2009) Whole Lung RegeneraDon Alleviates donor shortage problem XenotransplantaDon-organ is decellularized leaving only ECM behind as a "biological scaffold" Engineered matrix- arDficially engineered matrix is used as scaffold for re-cellularizaDon
24 hr PaDents own cells can be used to minimize risk of rejecDon
Song et al., Ann Thorac Surg. 2011 14 d AmnioDc Fluid Stem Cells (AFSC):
Novel tool to ameliorate inflammaDon and prevent fibrosis? Pluripotent (Carraro et al., 2008) (De Coppi et al., 2007) Inflammatory homing mechanism Decrease inflammatory cytokines in acute renal injury (Perin et al., 2010) Perin et al., 2010. DifferenDate to express Type II cell phenotypes in acute injury (Carraro et al., 2008)
JAMA. 2007;297:795. Intravenous InjecDon of AFSC shows localizaDon to the lung.
(AC): Mice injected with amnio=c fluid stem cells (AFSC) expressing luciferase. Ader 21 days AFSC were s=ll detected in the lung. (AC) present different levels of reten=on, A-maximum observed, C-minimum observed. (color scale is calibrated in arbitrary bioluminescence units). (D), the yellow arrow shows CM-Dil-labeled hAFSC (red) in an alveolar wall, corresponding with a type I pneumocyte stained for PDPN (green). (E), chromogenic in situ hybridiza=on for Y Chr also showed hAFSC (yellow arrow) in the alveolar wall posi=on. (F), immunohistochemistry for the an=gen F4/80 was performed to detect macrophage ac=vity (green). Carraro et al., 2008 Lung injury increases the level of retenDon of hAFSC Number of hAFSC in control mice and in mice aler oxygen versus naphthalene injury (n = 4) at 7, 15, and 40 days. RetenDon of hAFSC in mouse adult lung increased significantly aler injury (p < .001)
Carraro et al., 2008 AFSC and inflammaDon:
AFSC migrate to sites of inflammaDon CXCR4 present on surface of AFSC SDF-1 signaling up regulated in Naphthalene Injured Lung SuggesDve of mechanism involved in homing AFSC to site of injury in response to inflammaDon
Carraro et al., 2008 AFSC and lung injury:
Transplanted AFSC can express Type II phenotypes in vivo 15 days post hyperoxic injury AFSC double stain for pro- SPC and the Y-Chromosome Data indicates that in vivo, without the presence of addiDonal growth factors, AFSC can express Type II phenotypes Carraro et al., 2008 hAFSC are retained in the upper airways and express the Clara cell-specific marker CC10 Murine lungs damaged with 275 mg/kg naphthalene, and then transplanted with ~2.5 105 hAFSC administered intratracheally. Carraro et al., 2008 Key Points: when designing and evaluaDng your proposed therapy Does it work? What does it do? How can you improve it?
*Remember funcDonal assays are the "Holy Grail" Acknowledgements
Roger De Filippo, MD Sargis Sedrakyan, PhD Student Stefano Da Sacco, PhD Anna Milanesi MD/PhD Liron Shiri, Tech Astgik Petrosyan, PhD Student Valen=na Villani, PhD Student Ilenia Zanusso, PhD Student Dr. K. Lemley (CHLA/USC) Dr. D. Warburton (CHLA/USC) Lung
David Warburton, MD/DSc Gianni Carraro, PhD Sue Buckley, Tech Gianluca Turcatel, PhD Wei Shi, MD Barbara Driscoll, PhD ...
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- Fall '08
- Nephrology, Nephron, acute kidney injury, Renal cortex, AFSC