Harnessing Pluripotency

🧬 Excited to share our latest study now In Press: In this study led by rising star @sarahcolpitts.bsky.social, we show that IL-10⁺ #ILC2s prevent rejection and improve islet graft survival and function in humanized mouse models. Read more: doi.org/10.1016/j.aj... #Immunology #ImmunoSky #CellTherapy

PART 2 - the collecting system forms 🚰

Immunofluorescence images showing the changes in the combined UB and NPC culture from days 0-4. GFP and RET mark the UB and SIX1, SIX2 mark the NPCs. Nephrons induced into NPCs express JAG1 and LHX1 within 48 hours.

PART 1 - Setting up the kidney-forming niche in vitro 🔬

Using their previous protocols, they combined hPSC-UB 🌱 (RET+) and hPSC-NPCs🫘 (SIX1/2+). A simple media with only ROCK1/2 inhibitor and BMP inhibitor for 48 h was enough to induce epithelial vesicles (JAG1+/LHX1+) and a branching UB.

Schematic of how the ureteric bud and the metanephric mesenchyme combine to form the kidney and its collecting system

BACKGROUND 🧩

A kidney forms when two cell clusters meet:

• Metanephric Mesenchyme – contains NPCs that will become the filtration units/nephrons
• UB – branching duct that connects with NPCs/nephrons to drain them🚰

hPSCs can make both, but getting them fuse in vitro is what today’s paper tackles.

HIGHLIGHTS

• Optimized UB 🌱 and NPC🫘 co-culture system
• NPC and UB fusion happens in vitro🧪 and in vivo🐁
• UB fuses specifically with GATA3⁺ distal nephron segments
• Notch inhibition in NPCs boosts GATA3⁺ segments
• WNT+TGFβ+MAPK inhibition enhances AQP2⁺ in UB-derived collecting ducts (CD)

🙌 Thanks for following this kidney-organoid deep-dive!
What grabbed you most, and which papers or topics should Pluripotency unpack next?
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FINAL THOUGHTS:

- Important advances on methods to generate NPCs
- Could this become a new cell therapy in kidney disease?💉
- If VEGF-A is the key, do we really need kidney organoids, or can find another way to give it?
- More work needed to make functional, integrating kidney tissue from hPSCs! 👩‍🔬

SOME LIMITATIONS:

-All experiments done with a single source of hPSCs - limits generalizability
-NOD-SCID mice have multiple immune deficits, would NPCs behave similarly in "regular" mice (or humans)?👥
-Less than 15% of cells in the transplant were NPC-derived kidney cells. What were the rest?🤔

Top: Histology pictures showing effect of unmodified NPCs and VEGF-A deficient NPCs on kidney injury. Bottom: Bar graphs quantifying the effects of saline injection, unmodified NPCs and VEGF-A deficient NPCs on histologic features of kidney injury

PART 4: MECHANISM OF BENEFIT

Surprisingly, the NPCs did not integrate with the host kidney. Instead, they were found to produces VEGF-A after transplantation. VEGF-A hydrogels without NPCs could protect from AKI, and genetic inactivation of VEGF-A in NPCs greatly reduced their benefits.

A) Experimental schematic; B) Demonstration that NPC transplantation protects from increases in BUN and serum creatinine after aristocholic acid injection. C-D) Histology and bar graphs showing that NPC injection also protects from histologic measures of fibrosis in this model.

PART 3: TREATING KIDNEY DISEASE

Using a mouse🐁 model of AKI (cisplatin), they made the surprising finding that histologic tubular damage and serum creatinine can be improved by NPC injection and NOT terminal organoids. Similarly, NPCs could improve fibrosis in a model of CKD (aristocholic acid)

Bar plots showing enrichment of key NPC genes (SIX2, SALL1, HOXD11) in NPCs sorted using the novel marker c-MET.

Part 2: PURIFYING NPCs USING c-MET

One problem with hPSC technology is that cells don't always behave consistently. Planning for cases where cells don't differentiate to NPCs well, they found a protein specific to NPCs, "c-MET", that could be use to purify them using a technology called FACS.

Left: Images showing the effect of each factor on SIX2 and OSR1 expression. Right: Bar plot showing the effect of each factor on cell numbers.

PART 1: MAKING BETTER NPCs

Aiming to treat humans with kidney disease, they realized they would need to make tons of NPCs. So they first improved the method of growing these OSR1+SIX2+ NPCs in large numbers, using "CFY" media💧 containing:
C: Canonical Wnt agonist
F: FGF9
Y: ROCK1/2 inhibitor

Schematic showing how hPSCs differentiate into NPCs and then into kidney organoids

BACKGROUND:

Human pluripotent stem cells (hPSCs) can generate any tissue - including the kidney🫘. To do this, they must first specialize into nephron progenitors (NPCs), which are kidney-specific stem cells (can't make other organs).

The authors published on this before
e.g. tinyurl.com/4ufzv9pf

HIGHLIGHTS:

• A new culture medium (food for cells) helps nephron progenitors (NPCs) to grow in vitro
• An new protein ("c-MET") can be used to purify NPCs
• Transplanting NPCs attenuates acute (AKI) and chronic (CKD) kidney disease in mice
• VEGF-A mediates this

More details below ⬇️

Human iPSC–derived nephron progenitor cells treat acute kidney injury and chronic kidney disease in mouse models Expanded human iPSC–derived nephron progenitor cells secrete renoprotective factors that have therapeutic potential for AKI and CKD.

📣 NEW PAPER ALERT! 🧵
I love kidney organoids - tiny nephron clusters grown in the lab from pluripotent stem cells. But can they treat kidney disease? New data suggests they can (in mice)!
Let's dive in ⬇️
tinyurl.com/mr3sd6vu
#KidneyOrganoids #CellTherapy #ChronicKidneyDisease #AcuteKidneyInjury