Generation of hepato-biliary-pancreatic organoid from human pluripotent stem cells CURRENT STATUS: POSTED

Organogenesis is a complex and inter-connected process, orchestrated by multiple boundary tissue interactions. Here, we established the protocol of the continuous patterning of hepatic, biliary and pancreatic structures from a three-dimensional culture of human pluripotent stem cell (PSC). The boundary interactions between anterior and posterior gut spheroids differentiated from human PSCs enables autonomous emergence of hepato-biliary-pancreatic (HBP) organ domains in the absence of extrinsic factor supply. This anterior-poterior gut interaction protocol can be used to model the early human HBP organogenesis process in vitro .


Introduction
The hepato-biliary-pancreatic (HBP) anlage, which is demarcated by HHEX (Hematopoieticallyexpressed homeobox protein) and PDX1 (Pancreatic and duodenal homeobox 1) expression is specified at the boundary between the foregut-midgut.
Here, we leverage a three-dimensional differentiation approach using human pluripotent stem cells (PSCs) to specify gut spheroids with distinct regional identities comprised of both endoderm and mesoderm. We show that antero-posterior interactions recapitulate the foregut and the midgut boundary in vitro, modeling the inter-coordinated specification and invagination in the human hepatobiliary-pancreatic organoid (HBPO).

Maintenance of PSCs
Maintain the undifferentiated hPSCs on feeder-free conditions in StemFit (Ajinomoto Co., Inc.) or mTeSR1 medium (STEMCELL technologies) on plates coated with iMatrix-511 (Matrixome, Inc.) or Matrigel (Growth Factor Reduced; Corning Inc.) in an incubator with 5% CO 2 at 37°C. Dissolve the Matrigel (Growth Factor Reduced) in ice-cold DMEM/F12 at 1:30 dilution to coat an entire well of culture plate.

Differentiation of PSCs into anterior and posterior gut spheroid
Differentiation of hPSCs into definitive endoderm was induced using previously described methods 1,2 with modifications. Culture the cells in an incubator with 5% CO2 at 37°C. Change the culture medium at the same hour each day without DPBS wash.

Day -2
1) Dissociate colonies of hPSCs by Accutase into single cells and plate the cells with 100,000 -150,000 cells/cm 2 in mTeSR1 including 10µM Y27632 on Matrigel coated tissue culture plate (Corning). Prepare cells with at least 2 independent wells to generate anterior and posterior gut spheroids at the same time.

Day 7
At day 7, confirm that the differentiated cells start to come up from bottom of plate as shown in previous reports 1,2 .

8) Dissociate each of anterior or posterior gut cells into single cells by incubation with TrypLE Express
at 37°C and collect the each type of cells into 15 mL centrifuge tubes separately . 9) Centrifuge each tubes at 1000 rpm for 3 minutes and, after removing supernatant, re-suspend the pellets in Gut growth medium with 10 µM of Y-27632. 10) Plate the anterior or posterior gut cell suspensions on 96 well round bottom ultra-low attachment plate (Corning Inc.) at density of 10,000 cells/well. 11) Centrifuge the plate at 1000 rpm for 1 minute to gather cells and incubated at 37°C for 24 hours to form spheroid.

Day 8
12) Mix the generated single anterior spheroid and posterior spheroid on 96 well round bottom ultralow attachment plate in gut growth medium without Y27632.
13) Centrifuge the plate at 1000 rpm for 1 minute to gather spheroids and incubated at for 24 hours to form fused boundary spheroids (anterior-posterior gut spheroid; A-P spheroids).

Day 9 -13
14) Pickup an A-P spheroid into centrifuge tube by mechanical pipette with wide bore tip. Re-suspend the A-P spheroid in 20 -30 µL/spheroid/well of 100 % Matrigel (Phenol Red-Free) on ice and plate it on 24 well tissue culture plate (VWR international) to make Matrigel drop. 15) Incubate the drop in CO 2 incubator at 37°C for 5 minutes. · Confirm the cell confluency at day 0 is around 90 %.
· Make sure that each aliquots of small molecule or recombinant protein should be reconstituted and stored appropriately according to manufacture's document.
· Do not store the medium in refrigerator for more than a week after adding small molecules, recombinants, serum or supplements which are not recommended to keep in refrigerator .
· Check the concentration of each component in differentiation medium.
· Make sure that all medium change was performed at the same hour each day.

Day 8
Problem: Gut spheroids are not formed Solution: · Use TrypLE Express for dissociation at day 7, not Accutase, EDTA or Trypsin. 9 · Make sure that 10 µM of Y-27632 was added in medium at day 7. · Make sure that the seeding cell number was appropriate at day 7. · Centrifuge the plate at 1000 rpm for 1 minute before starting culture at day 7. · Check the Solutions described in above for the problem of day 7.

Anticipated Results
The boundary interactions between anterior and posterior gut spheroids differentiated from human pluripotent stem cells enables autonomous emergence of hepato-biliary-pancreatic (HBP) organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factor supply.
This enables the subsequent study for early morphogenesis of HBP subdomains, and for generating inter-connected, multi-organ structures within personalized human model systems for organogenesis and disease in vitro.