Poster U13 - A microfluidic patterned endoderm tube model using human pluripotent stem cells

Introduction: During embryogenesis, gastrulating mesendoderm cells migrate and form the definitive endoderm layer, and transform into a tubular endoderm structure, with an organized anterior-posterior (A-P) and dorsal-ventral (D-V) pattern under the control of morphogen gradients and mechanical forces from adjacent tissues. In recent years, human pluripotent stem cell (hPSC)-based endodermal organoids are emerging, providing useful experimental tools to advance molecular and cellular understanding of endodermal organ development and related diseases. However, most of the existing endodermal organoid models could manifest only certain parts of gastrointestinal (GI) developmental events, and they are not capable of mimicking the structurally organized entire GI patterning along the A-P axis in a 3D tubular geometry. Thus, current endodermal organoid models remain ineffective in studying regional specification and inter-tissue communications during GI development.

Microfluidic gradient-based hPSC models have recently shown promise in studying different early embryogenesis events. Such microfluidic tools provide controllable mechano-biological orchestrated systems that can mimic mechanical sensing and morphogen gradients during natural development. Here, we combine bioengineering tools, including microfluidic gradients, to recapitulate the whole early endoderm tube development. Characterized by several apical-basal and other membrane markers, we build a structurally organized tubular endodermal system. We further show that this tubular endodermal structure exhibits well-delineated A-P patterning. Our work generates a whole endoderm tube development model that mimics tubular structure formation and patterning process, and gives insight into dynamic regulation of early endoderm development.

Materials and

Methods: Human ESC lines
Human embryonic stem cell (hESC) line H1 was used in this study. All protocols with hPSCs have been approved by the Human Pluripotent Stem Cell Research Oversight Committee at the University of Michigan. Cells were maintained on feeder-free cultures.

Differentiation of definitive endoderm cells
On Day 0, H1 hESCs were dissociated by Accutase and suspended as single cells, before being plated on 6-well plates pre-coated with 1% Geltrex. On Day 1, culture medium was switched to RPMI1640 supplemented with 100 ng/mL Activin A and 50 ng/mL BMP4. Cells in the following two days were treated with Activin A plus 0.2% (Day 2) and 2% (Day 3) dFBS. On Day 4, definitive endoderm cells were harvested from culture plates and were injected into microfluidic devices as described below.

Microfluidic device fabrication and development
To fabricate microfluidic devices, rectangular Geltrex adhesive islands (length: 2 mm; width: 100 µm) were printed onto a coverslip using microcontact printing with a PDMS stamp. After micropatterning, a PDMS structural layer was then attached onto the coverslip, with printed Geltrex islands aligned with the central channel of the PDMS structural layer. Dissociated single definitive endoderm cells derived from hESCs were then loaded into the central channel and allowed to adhere to Geltrex islands. One hour after cell seeding, floating human definitive endoderm cells not attached to Geltrex islands were flushed away gently. On Day 5, 100% Geltrex was loaded into the central channel, and endoderm tube induction medium was added to the two reservoirs.

Results, Conclusions, and Discussions: hPSCs was treated with Activin A for 3 days to generate FOXA2+SOX17+ definitive endoderm (DE) cells. Using our microfluidic device, we showed that these DE cells could successfully form organized tubular structures containing a single, central lumen on Day 9, evidenced by immunostaining for apical marker ZO-1 and E-cadherin, mimicking the in vivo endoderm tube features. A well-patterned foregut-hindgut axis (A-P axis) was generated by precisely controlling WNT, BMP, FGF, and RA signaling using microfluidic gradients. Foregut marker SOX2 and mid/hindgut marker CDX2 showed upregulated expression in anterior and posterior regions of the endoderm tube, respectively. PDX1 further showed high expression in the middle part of the endoderm tube, mimicking the in vivo pattern. These results together indicated a well-patterned endoderm tube with a regionalized foregut-midgut-hindgut axis was generated. Together, this work reports a promising hESC-derived model to study dynamic development of the endoderm tube and its patterning process, which offers a useful experimental platform for both fundamental studies and translational applications including drug screening and disease modeling.

Acknowledgements (Optional):