Stem Cells inKidney Disease:

Where Are We Now?

Berliner Dialyseseminar5. Dezember 2015

Albert Q. Lam, MD

Associate PhysicianBrigham and Women’s Hospital

Harvard Medical School

Disclosures

• None

Regenerative medicine

Disease modeling

Cell transplantation

Drug development

Bioengineering

Polycystic kidney disease

Alport’s syndrome

FSGS

• Two basic components:– Cells

• Starting substrate• Stimulate regeneration• Promote vascularization

– Biomaterials• Natural or synthetic matrix• Provide spatial environment

to restore tissue structure and function

• Attract/suppport cells

Bioengineering a Kidney

Human Pluripotent Stem Cells

Can differentiate into cells and tissues of all three germ layers of the embryo

Types:Embryonic stem cellsInduced pluripotent stem cells (iPS cells)Embryonal carcinoma cellsEmbryonic germ cells

PSC

Ectoderm (nervous system, epidermal tissues)

Mesoderm (muscle, bone, blood, heart, kidneys)

Endoderm (lungs, stomach, pancreas, GI tract)

Embryonic Stem (ES) Cells

Modified from Landry and Zucker J Clin Invest 2004

Specialized cells:

CardiomyocytesHepatocytes

Pancreatic cellsIntestinal cells

NeuronsMuscle cellsKidney cells

Induced Pluripotent Stem (iPS) Cells

OCT4SOX2KLF4

C-MYC

Human iPS Cell Lines from Patients with Kidney Disease

Lam, Freedman, and Bonventre Semin Nephrol 2014

Kidney Organogenesis

• The kidneys are derived from the mesoderm germ layer (intermediate mesoderm)

Metanephric mesenchyme

Ureteric bud

SIX2+ nephron progenitor cells“Kidney stem cells”

http://design.ninabosanac.com/portfolio-item/kidney-development-illustrations/

Origin of the Metanephric Mesenchyme

Taguchi et al. Cell Stem Cell 2014

Human PSCs into Kidney Cells

2000-2010

• Schuldiner PNAS 2000• Batchelder Differentiation 2009• Lin Stem Cells Dev 2010

2012-2015

• Song PLoS One 2012• Narayanan Kidney Int 2013• Mae Nat Commun 2013• Xia Nat Cell Biol 2013• Taguchi Cell Stem Cell 2013• Takasato Nat Cell Biol 2013• Lam JASN 2013• Araoka PLoS One 2014• Kang PLoS One 2014• Kandasamy Sci Rep 2015• Imberti Sci Rep 2015• Toyohara Stem Cells Transl

Med 2015• Takasato Nature 2015• Freedman Nat Commun 2015• Morizane Nat Biotechnol 2015

Recapitulating Metanephric Development

PSCsLate mid

primitive streak

Posteriorintermediate mesoderm

Metanephricmesenchyme

Defining the Origin of the Metanephric Mesenchyme

To generate the primitive streak cells that will give rise to MM:

• Define the anterior-posterior position along the primitive streak(mid-streak)

• Define the timing of migration out of the primitive streak(late stage)

Extended treatment with high -dose CHIR99021 induces late mid -primitive streak

Day 4

Morizane et al. Nat Biotechnol 2015

Activin induces posterior IM differentiation

Morizane et al. Nat Biotechnol 2015

Day 7

FGF9 induces metanephric mesenchyme

Day 9

Morizane et al. Nat Biotechnol 2015

SIX2+ NPCs spontaneously differentiate into renal vesicles

Morizane et al. Nat Biotechnol 2015

FGF9 + CHIR induce renal vesicles

Day 14

Morizane et al. Nat Biotechnol 2015

Renal vesicles spontaneously form nephron structures in 2D culture

Day 21

Morizane et al. Nat Biotechnol 2015

Renal vesicles spontaneously form nephron structures in 2D culture

Day 21

Morizane et al. Nat Biotechnol 2015

Nephron structures in 2D cultureDas Bild kann zurzeit nicht angezeigt werden.

Days 21-28

Morizane et al. Nat Biotechnol 2015

Nephron progenitor cells form spherical aggregates in 3D suspension culture

Morizane et al. Nat Biotechnol 2015

3D organoids contain multi-segmented nephron structures

Morizane et al. Nat Biotechnol 2015

3D organoids contain multi-segmented nephron structures

Morizane et al. Nat Biotechnol 2015

3D organoids contain multi-segmented nephron structures

Morizane et al. Nat Biotechnol 2015

Electron microscopy of 3D nephron organoids

3D OrganoidsHuman Kidney

Morizane et al. Nat Biotechnol 2015

Modeling development with organoids

Morizane et al. Nat Biotechnol 2015

Notch inhibition

2D organoids

DAPT impairs proximal tubule formation

Morizane et al. Nat Biotechnol 2015

DAPT

Control

3D organoids

Modeling kidney toxicity with organoids

3D organoid

Gentamicin x 48 hours

Modeling kidney toxicity with organoids

Summary

• By mimicking the critical developmental stages of nephrogenesis, human PSCs can be efficiently differentiated into multipotent nephron progenitor cells (NPCs)

• NPCs can be induced to form renal vesicles that spontaneously form multi-segmented nephron structures in 2D and 3D organoids

• Nephron organoids can be used to model kidney development and toxicity

Human PSCs into Kidney Cells

2000-2010

• PAX2+vimentin+ cells• WT1+PAX2+ cells

2012-2015

• Proximal tubule-like cells• Podocyte-like cells• Intermediate mesoderm cells• Ureteric bud progenitor cells• Nephron progenitor cells• 2D and 3D kidney organoids

Applications

• Modeling kidney toxicity• Modeling kidney development• Modeling kidney disease• Modeling kidney physiology• Repair after AKI

AcknowledgementsBrigham and Women’s Hospital•Joseph Bonventre, MD, PhD•Ryuji Morizane, MD, PhD•Todd Valerius, PhD•Seiji Kishi, MD•Vanesa Bijol, MD

University of Washington•Benjamin Freedman, PhDNorthwestern University•Jason Wertheim, MD, PhD