EB size mediated Cardiomyogenesis and Vasculogenesis

DATE CREATED:

ACTION PLAN

PROBLEMS IDENTIFIED (PI) / OUTSIDE SKILL REQUIRED (OSR) / RESOLVED (R)

PAPER TITLE

Embryoid Body Size Dependant Embryonic Stem Cell Fate Specification Into Cardiogenesis And Angiogenesis Via Differential Wnt5a and Wnt11 Noncanonical Pathway.

A) BACKGROUND:

Embryonic stem (ES) cells have great potential for the cell-based therapy and regenerative medicine. ES cells are characterized by two unique properties, namely their “unlimited self-renewal capacity” and “pluripotency”, which enables the ability to generate various fetal and adult cell types in vivo and in vitro. These properties make ES cells, potentially, an invaluable cell source for a variety of tissue-engineering and clinical applications. Currently, widely accepted one of major approaches has been reported to induce specific lineage differentiation from ES cells through embryoid body (EB) formation. When ES cells are cultured by several methods such as hanging drop culture and suspension culture, they form three-dimensional (3D) aggregate intermediates, called EBs, which initiate developmental process and generate derivatives of three primary germ layers (i.e. ectoderm, mesoderm, and endoderm). For the ES cell differentiation into cardiomyogenic lineage, suspension culture and EB hanging drop culture methods have been widely used. EBs are generally formed by suspension of ES cells in bacteriological dishes in the absence of adhesion to the culture dish (using the appropriate EB formation medium), resulting in the spontaneously formed three germ layers that provide the pool of progenitors for the subsequent cardiomyogenic differentiation. The generation of cardiomyogenic cells is achieved by replating the EBs in monolayer cultures, following EB formation for proper period, and differentiation with the appropriate supplements. In those approaches, the duration of EB formation and medium supplements were generally considered as parameters to induce cardiomyogenic differentiation because conventional culture methods (i.e. hanging drop culture and suspension culture) have limitations to control other papameters such as the inability to control homogeneous EB size. Despite the progress made for EB mediated lineage specific differentiation by controlling EB formation period and the increased understanding of the biological mechanisms of growth factors or other supplements on ES cells differentiation, the challenge for controlling ES cell differentiation with other parameters is still needed to be addressed. In order to address this challenge, we hypothesized that three germ layer formation and further lineage specific differentiation could be affected by the size of ES cell colony and EBs. Recently, several attempts have been made to study difference in differentiation of ES cells by controlling the ES cell aggregate and EB size using micropatterning and micro contacting techniques, but they might be limited to maintain initial cell colony/EB size and homogenous culture environment throughout whole culture period, regarding the EB enlargement, agglomeration and the interaction between EBs in transferred suspension culture system. Hence, in this paper, we generated microfabricated poly(ethylene glycol) (PEG) hydrogel microwells with various diameters (150, 300, and 450 µm) as we have previously developed in order to generate and maintain homogenous EB size, and demonstrated the shear-protective microwell arrays using the computational simulation. ES cells formed homogenous EBs with different sizes within hydrogel microwell arrays throughout whole culture period and EBs were easily retrieved from microwells. Furthermore, their early differentiation into three germ layers and further ES cell fate specification into cardiogenesis and angiogenesis were analyzed and characterized by using immnostaining, RT-PCR, and siRNA transfection, and finally the mechanism to determine ES cell fate specification was investigated.

B) Hypothesis

Generally, the duration of embryoid body formation has been accepted as most important parameter to control embryonic stem cell differentiation through embryoid body formation. However, supposing the increase of embryoid body size in time course manner, is really the embryonic stem cell differentiation within embryoid body time dependant? or embryoid body size depedant?

C) SPECIFIC AIMS

AIM 1 -

AIM 2 -

D) GENERAL EXPERIMENTAL APPROACH

E) DESIGN PITFALLS AND ALTERNATIVES

F) ANTICIPATED FIGURES FOR PAPER or when you have data, FIGURES FOR PAPER

G) FUTURE DIRECTIONS