Organ Regeneration Based on Developmental Biology

205

Author,Year

CultureMethod

Derivation of IntermediateMesoderm (IM) via PosteriorPrimitive Streak (PPS)

Derivation of MetanephricMesenchyme (MM) and/orUreteric Bud (UB)

Endpoints and markers by

immunofluorescence

Notes

GlomP

TD

TC

DE

ndoS

troma

Mae et al.(2013)

Monolayer

PPS:

3 days CHIR99021 + Activin A

IM:

8 days CHIR99021 + BMP7

MM

: 7 days CHIR99021, BMP7

++

Generated an OSR1-GFP reporter line toenable high throughput growth factorsscreening for IM induction.

Taguchiet al.(2014)

EmbryoidBody

Epiblast:

2 days Activin A

PPS:

2 days CHIR99021, BMP4

Posterior nascent mesoderm:

4 days

CHIR99021, BMP4Posterior IM:

2 days Activin A, BMP4,

CHIR99021, RA

MM:

3 days CHIR99021, FGF9

++

+

MM was derived under fully definedconditions but culture of organoid en

d

points was by culture with dorsal spinalcord.

Lam et al. (2014a, b)

Monolayer

PPS:

2 days CHIR99021

IM:

4 days FGF2 + RA

MM:

3 days FGF9 + activin A+

IM differentiated into LTL+, N-cadherin+ PT cells with removal of FGF2and RA and 1 day CHIR99021.

Takasato et al.(2014)

Monolayer

PS:

2 days CHIR99021

IM:

4 days FGF9 + Heparin

MM and UB:

6 days FGF9 + Heparin

then 6 days no growth factors.

++

++

Early podocytes colocalising WT1+ an

d

synaptopodin+ with PCR evidence forupregulation of podocyte genes.

Araoka et al. (2014)

Monolayer

PPS:

2 days CHIR99021 + Activin A

IM:

4 days AM580 or TTNPB

MM and UB:

8 days Wnt3a + BMP7

++

+?

Also found CHIR99021 alone can giverise to PPS. TTNPB protocol was utilisedfor the differentiation of end point cultures.

Imberti et al. (2015)

Monolayer

PPS/IM:

6 days RA + PI3K inh + RhoA

inh + 2 days activin A

MM:

FGF2 + BMP7 + GDNF

++

Integration of human renal progenitors intocisplatin injured mouse nephrons wa

s

observed.

Morizane et al.(2015)

Monolayerthen 3D

Late PS:

4 days CHIR99021 ± noggin

Posterior IM:

3 days activin A

MM:

2 days FGF9

Self-organising nephrons:

2 days

FGF9 + CHIR99021 then 3 daysFGF9 then no growth factors.

++

+a

3D culture performed by replating cells onday 10 to ultra-low-attachment, roundbottom 96-well plates and in suspensionculture

.

Freedman et al.(2015)

Spheroid

Epiblast spheroid:

2 days sandwiched

between Matrigel in mTeSR1 mediumMesoderm:

1.5days CHIR99021

Self-organising nephrons:12 days B27 supplemen

t

++

++

Generated epiblast spheroids in Matrigelsandwich culture, prior to differentiation.CD31+/vWF+ endothelium also present

.

Takasato et al.(2015)

Monolayerthen 3D

PPS:

4 days CHIR99021

Ant and Post IM:

3d FGF9 + Heparin

Organoid:

1 hour CHIR99021,

then 5 days FGF9 + Heparin,then 7 days no growth factors.

++

++

++

First report of endothelial progenitors(CD31+, SOX17+) with lumen formation,observed invading glomeruli.

Table 11.1

Fully defined protocols for the in vitro directed differentiation of human iPSC to multisegmented nephron fate

IM

intermediate mesoderm,

PPS

posterior primitive streak,

RA

retinoic acid,

Glom

glomerular structures,

PT

proximal tubules,

DT

distal tubule,

CD

collecting

duct,

Endo

endothelium

aPersonal communication

11 Recapitulating Development to Generate Kidney Organoid Cultures