Directing Human Induced Pluripotent Stem Cell-Derived Embryonic-like Organoid with 3D Biomimetic Cryogel Mechanical Microenvironment for Neural Induction.

Research Project

Project/Area Number	22K20642
Research Category	Grant-in-Aid for Research Activity Start-up
Allocation Type	Multi-year Fund
Review Section	0701:Biology at molecular to cellular levels, and related fields
Research Institution	Institute of Physical and Chemical Research
Principal Investigator	Vadivelu RajaKumar 国立研究開発法人理化学研究所, 開拓研究本部, 基礎科学特別研究員 (60963543)
Project Period (FY)	2022-08-31 – 2024-03-31
Project Status	Granted (Fiscal Year 2022)
Budget Amount *help	¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000) Fiscal Year 2023: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000) Fiscal Year 2022: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Keywords	hIPSCs / Gastruloid / Cryogel / Mechanobiology / Neural Induction / Neuromesoderm / organogenesis / pluripotent / Embryonic Organoid / Neuromesodermal
Outline of Research at the Start	The study will generate a mechanical microenvironment to direct iPSCs to generate embryo-like organoid called gastruloid without the use of exogenous chemical stimulation. The overall purpose to understand morphogenetic events taking place during tissue growth and elucidate neuromesodermal tissue specification.
Outline of Annual Research Achievements	We aim to investigate how using cryogel sandwich model by embedding cell clusters with layer-by-layer cryogel could enhance mechanical constraints. First, we studied cryogel`s topology by using Hirox, and SEM microscope analysis revealed that cryogel surface topology consists of voids that appear as hill-valley-like structures composed of pockets like void space which can trap microbeads. Thus, cryogel facilitates spontaneous cell cohesion, leading to spherical-shaped aggregates within 24h. After cryogel embedding the aggregates acquire a teardrop-like shape. Eventually, on day 3 these aggregates showed up ovoid shape which resembles a symmetrical breaking process. Day 4 showed polarized aggregates with the posterior axis.
Current Status of Research Progress	Current Status of Research Progress 3: Progress in research has been slightly delayed. Reason We focus on establishing methodological development to elucidate cryogel properties on stimulating mechanical input and impact iPSCs aggregation. For this purpose, we optimized cryogel fabrication at different freezing times and evaluate the topological features accordingly. The second optimization is determining the appropriate culture condition. We first optimized the 2D cell culture regime by using Matrigel and vitronectin. Along, with this we opt to find the suitable media either using Stem fit or E8. After establishing 2D cell culture regime we then determine the suitable growth condition of 3D culture. Then we tested the effect of ROCK inhibitor on the viability of the 3D culture. Also, include shifting from stem fit during 2D culture to E8 at the 3D growth phase.
Strategy for Future Research Activity	Plan 1: The progressing research plan is to optimize the adequate and reproducible method in culturing iPSCs aggregates and how embedment method gives axial morphogenesis. Plan 2: We will assess the impact of cryogel embedding on pluripotent exit and evaluate the localization of differentiated cells in the establishment of markers responsible for gastrulation. Particularly, the capacity to self-organize into a neural fate which is possibly rudimentary along the A-P axis. Next, we aim the examine the transition of core pluripotency protein Sox2+ cells to prime state pluripotency marked by Otx2 which is in the state of the stable epiblast. Later, we aim to identify mutually exclusive patterns between Otx2 and T-Bra with Sox2 that could occur.