Engineering system platforms for human stem cell maintenance and differentiation

Funding Type: 
SEED Grant
Grant Number: 
RS1-00381
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Embryoid body (EB) formation is a potent method for differentiating human embryonic stem cells (hESCs). EBs formed on commercially available low attachment plates, however, form asynchronously, tend to vary in size and aggregate, making the differentiation process heterogeneous and uncontrollable. The overall goal of this research project is to establish the conditions required to regularize the formation of EBs for multiple hESC lines, thereby obtaining controlled differentiation to the lineages or cells of interest. Our approach is based on a nano/micro technology platform in which new materials are integrated with microfluidic devices and biomarker sensors. The platform we develop from these components will lead to robust, programmable methods for growing EBs that are far superior to current EB formation protocols. We plan to use this technology platform in combination with a feedback control scheme of the type used to guide complex engineering systems to improve differentiation efficiency. The project consists of two technical aims: 1. Develop a nano/micro technology platform that uses non-adherent surfaces, micromachined three-dimensional architectures and microfluidic chemical delivery actuators to provide a controlled environment for massively parallel growth of uniformly-sized EBs. 2. Apply this technology platform to endoderm differentiation with the objective of improving the differentiation efficiency and yield of endoderm lineage, especially insulin-secreting pancreatic cells. The development of a robust technology platform with programmable microfluidics and feedback control that removes the variability of EB size and shape offers the promise of achieving high throughput, scalable systems for therapeutic applications.
Statement of Benefit to California: 
Human Embryonic Stem Cells (hESCs) are promising candidates for tissue replacement therapies for several debilitating pathologies including diabetes, leukemia, and heart diseases, as well as for advancing regenerative medicine research. The formation of spherical aggregates known as embryoid bodies (EBs) is a critical intermediate in the differentiation of hESC into specific cell types needed for these therapies. Unfortunately, current EB processing methods provide low yield, inconsistent sizes, and unpredictable cell types. Achieving uniformly sized EBs with synchronized differentiation kinetics requires new tools to manage and control the environment of hESCs during the EB development stage. In this project, our stem cell researcher team leverages expertise in material engineering and nanotechnology to create robust, repeatable and programmable processing methods that will guide EB formation, growth, and differentiation. These new methods will be superior to current protocols because of the novel engineering techniques capable of monitoring and manipulating at the level of single cell and single embryoid body. This project will yield technology which will remove the variability of EB size and shape and offer the promise of achieving high throughput, scalable systems for therapeutic applications. This work will be of great importance in furthering the state of California’s interest in stem cell biology and regenerative medicine. Specifically, in the near term, the success of our strategy would lead to advances in differentiating hESCs toward insulin producing cells derived from the endoderm. Our work will provide valuable insight into the signaling cues responsible for this differentiation process. Ultimately, the ability to cultivate these cells in a controlled and scalable process would revolutionize the management of patients with diabetes mellitus which is expected to afflict 340 million US citizens by the year 2030. Secondly, the growth of industries interested in the development of hESC based therapies is expected to rise substantially in California given the state’s efforts to promote stem cell research. Translating this work to production scales is necessary and will be enabled through the development of the research tools that we propose.
Progress Report: 
  • CIRM Grant – Public Abstract:
  • Non-invasive imaging techniques for an in vivo tracking of transplanted stem cells offer real-time insight into the underlying biological processes of new stem cell based therapies, with the aim to depict stem cell migration, homing and engraftment at organ, tissue and cellular levels. We showed in previous experiments, that stem cells can be labeled effectively with contrast agents and that the labeled cells can be tracked non-invasively and repetitively with magnetic resonance imaging (MRI) and Optical imaging (OI). The purpose of this study was to apply and optimize these labeling techniques for a sensitive depiction of human embryonic stem cells (hESC) with OI and MRI.
  • Experimental Design: hESC were labeled with various contrast agents for MRI and OI, using a variety of labeling techniques, different contrast agent concentrations and different labeling intervals (1h – 24h). The cellular contrast agent uptake was proven by mass spectrometry (quantifies the iron oxides) and fluorescence microscopy (detects fluorescent dyes). The labeled hESC underwent imaging studies and extensive studies of their viability and ability to differentiate into specialized cell types.
  • Imaging studies: Decreasing numbers of 1 x 10^5 - 1 x 10^2 contrast agent-labeled hESC and non-labeled controls were evaluated with OI and MRI in order to determine the best contrast agent and labeling technique as well as the minimal detectable cell number with either imaging technique. In addition, samples of hESC were investigated with OI and MRI at 1 min, 2 min, 5 min, 1h, 2h, 6h, 12h, 24h and 48 h in order to investigate the stability of the label over time. Viability and differentiation assays of the hESC were performed before and after the labeling procedure in order to prove an unimpaired viability and function of the labeled cells.
  • Results: The FDA-approved contrast agents ferumoxides and indocyanine green (ICG) provided best results for MR and optical imaging (OI) applications. The cellular load with these labels was optimized towards the minimal concentration that allowed for detection with MR and OI, but did not alter cell viability or differentiation capacity. The ferumoxides and ICG-labeled hESCs as well as stem cell derived cardiomyocytes and chondrocytes provided significantly increased MR and OI signal effects when compared to unlabeled controls. ICG labeling provided short term labeling with rapid excretion of the label from the body while ferumoxides labeling allowed for cell tracking over several weeks.
  • Significance: The derived data allowed to establish and optimize hESC labeling with FDA approved contrast agents for a non-invasive depiction of the labeled cells with MR and OI imaging techniques. Our method is in principle readily applicable for monitoring of hESC -based therapies in patients and allows for direct correlations between the presence and distribution of hESC-derived cells in the target organ and functional improvements. The results of this study will be the basis for a variety of in vivo applications and associated further grant applications.

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