New research findings show that embryonic stem cells unable to fully compact the DNA inside them cannot complete their primary task: differentiation into specific cell types that give rise to the various types of tissues and structures in the body.

Researchers from the Georgia Institute of Technology and Emory University found that chromatin compaction is required for proper embryonic stem cell differentiation to occur. Chromatin, which is composed of histone proteins and DNA, packages DNA into a smaller volume so that it fits inside a cell. 

A study published on May 10, 2012 in the journal PLoS Genetics found that embryonic stem cells lacking several histone H1 subtypes and exhibiting reduced chromatin compaction suffered from impaired differentiation under multiple scenarios and demonstrated inefficiency in silencing genes that must be suppressed to induce differentiation.

“While researchers have observed that embryonic stem cells exhibit a relaxed, open chromatin structure and differentiated cells exhibit a compact chromatin structure, our study is the first to show that this compaction is not a mere consequence of the differentiation process but is instead a necessity for differentiation to proceed normally,” said Yuhong Fan, an assistant professor in the Georgia Tech School of Biology.

Fan and Todd McDevitt, an associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, led the study with assistance from Georgia Tech graduate students Yunzhe Zhang and Kaixiang Cao, research technician Marissa Cooke, and postdoctoral fellow Shiraj Panjwani.

The work was supported by the National Institutes of Health’s National Institute of General Medical Sciences (NIGMS), the National Science Foundation, a Georgia Cancer Coalition Distinguished Scholar Award, and a Johnson & Johnson/Georgia Tech Healthcare Innovation Award.

To investigate the impact of linker histones and chromatin folding on stem cell differentiation, the researchers used embryonic stem cells that lacked three subtypes of linker histone H1 -- H1c, H1d and H1e -- which is the structural protein that facilitates the folding of chromatin into a higher-order structure. They found that the expression levels of these H1 subtypes increased during embryonic stem cell differentiation, and embryonic stem cells lacking these H1s resisted spontaneous differentiation for a prolonged time, showed impairment during embryoid body differentiation and were unsuccessful in forming a high-quality network of neural cells.

“This study has uncovered a new, regulatory function for histone H1, a protein known mostly for its role as a structural component of chromosomes,” said Anthony Carter, who oversees epigenetics grants at NIGMS.  “By showing that H1 plays a part in controlling genes that direct embryonic stem cell differentiation, the study expands our understanding of H1’s function and offers valuable new insights into the cellular processes that induce stem cells to change into specific cell types.”

During spontaneous differentiation, the majority of the H1 triple-knockout embryonic stem cells studied by the researchers retained a tightly packed colony structure typical of undifferentiated cells and expressed high levels of Oct4 for a prolonged time. Oct4 is a pluripotency gene that maintains an embryonic stem cell’s ability to self-renew and must be suppressed to induce differentiation.

“H1 depletion impaired the suppression of the Oct4 and Nanog pluripotency genes, suggesting a novel mechanistic link by which H1 and chromatin compaction may mediate pluripotent stem cell differentiation by contributing to the epigenetic silencing of pluripotency genes,” explained Fan. “While a significant reduction in H1 levels does not interfere with embryonic stem cell self-renewal, it appears to impair differentiation.”

The researchers also used a rotary suspension culture method developed by McDevitt to produce with high efficiency homogonous 3D clumps of embryonic stem cells called embryoid bodies. Embryoid bodies typically contain cell types from all three germ layers -- the ectoderm, mesoderm and endoderm -- that give rise to the various types of tissues and structures in the body. However, the majority of the H1 triple-knockout embryoid bodies formed in rotary suspension culture lacked differentiated structures and displayed gene expression signatures characteristic of undifferentiated stem cells.

“H1 triple-knockout embryoid bodies displayed a reduced level of activation of many developmental genes and markers in rotary culture, suggesting that differentiation to all three germ layers was affected.” noted McDevitt.  

The embryoid bodies also lacked the epigentic changes at the pluripotency genes necessary for differentiation, according to Fan.

“When we added one of the deleted H1 subtypes to the embryoid bodies, Oct4 was suppressed normally and embryoid body differentiation continued,” explained Fan. “The epigenetic regulation of Oct4 expression by H1 was also evident in mouse embryos.”

In another experiment, the researchers provided an environment that would encourage embryonic stem cells to differentiate into neural cells. However, the H1 triple-knockout cells were defective in forming neuronal and glial cells and a neural network, which is essential for nervous system development. Only 10 percent of the H1 triple-knockout embryoid bodies formed neurites and they produced on average eight neurites each. In contrast, half of the normal embryoid bodies produced, on average, 18 neurites.

In future work, the researchers plan to investigate whether controlling H1 histone levels can be used to influence the reprogramming of adult cells to obtain induced pluripotent stem cells, which are capable of differentiating into tissues in a way similar to embryonic stem cells.

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under award number GM085261 and the National Science Foundation under award number CBET-0939511. The content is solely the responsibility of the principal investigators and does not necessarily represent the official views of the NIH or NSF.

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Stem cell technologies have been proposed for cell-based diagnostics and regenerative medicine therapies. However, being able to make stem cells efficiently develop into a desired cell type -- such as muscle, skin, blood vessels, bone or neurons -- limits the clinical potential of these technologies.

New research presented on June 16, 2011 at the annual meeting of the International Society for Stem Cell Research (ISSCR) shows that systematically controlling the local and global environments during stem cell development helps to effectively direct the process of differentiation. In the future, these findings could be used to develop manufacturing procedures for producing large quantities of stem cells for diagnostic and therapeutic applications. The research is sponsored by the National Science Foundation and the National Institutes of Health.

"Stem cells don't make any decisions in isolation; their decisions are spatially and temporally directed by biochemical and mechanical cues in their environment," said Todd McDevitt, director of the Stem Cell Engineering Center at Georgia Tech and an associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University. "We have designed systems that allow us to tightly control these properties during stem cell differentiation, but also give us the flexibility to introduce a new growth factor or shake the cells a little faster to see how changes like these affect the outcome."

These systems can also be used to compare the suitability of specific stem cell types for a particular use.

"We have developed several platforms that will allow us to conduct head-to-head studies with different kinds of stem cells to determine if one type of stem cell outperforms another type for a certain application," said McDevitt, who is also a Petit Faculty Fellow in the Institute for Bioengineering and Bioscience at Georgia Tech.

Many laboratory growth methods allow stem cells to aggregate in three-dimensional clumps called "embryoid bodies" during differentiation. McDevitt and biomedical engineering graduate student Andres Bratt-Leal incorporated biomaterial particles directly within these aggregates during their formation. They introduced microparticles made of gelatin, poly(lactic-co-glycolic acid) (PLGA) or agarose and tested their impact on the assembly, intercellular communication and morphogenesis of the stem cell aggregates under different conditions by varying the microsphere-to-cell ratio and the size of the microspheres.

The researchers found that the presence of the biomaterials alone modulated embryoid body differentiation, but did not adversely affect cell viability. Compared to typical delivery methods, providing differentiation factors -- retinoic acid, bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) -- via microparticles induced changes in the gene and protein expression patterns of the aggregates.

By including tiny magnetic particles into the embryoid bodies during formation, the researchers also found they could use a magnet to spatially control the location of an aggregate and its assembly with other aggregates. The magnetic particles remained entrapped within the aggregates for the duration of the experiments but did not adversely affect cell viability or differentiation.

"With biomaterial and magnetic microparticles, we are beginning to be able to recreate the types of complex geometric patterns seen during early development, which require multiple cues at the same time and the ability to spatially and temporally control their local presentation," noted McDevitt.

While microparticles can be used to control differentiation by regulating the local environment, other methods exist to control differentiation through the global environment. Experiments by McDevitt and biomedical engineering graduate student Melissa Kinney have demonstrated that modulating hydrodynamic conditions can dictate the morphology of cell aggregate formation and control the expression of differentiated phenotypic cell markers.

"Because bioreactors typically impose hydrodynamic forces on cells to cultivate large volumes of cells at high density, our use of hydrodynamics to control cell fate decisions represents a novel, yet simple, principle that could be used in the future for the scalable efficient production of stem cells," added McDevitt.

Technologies capable of being directly integrated into bioprocessing systems will be the best choice for manufacturing large batches of stem cells, he noted. In the future, the development of multi-scale techniques that combine different levels of control -- both local and global -- to regulate stem cell differentiation may help the translation of stem cells into viable clinical therapies.

This project is supported by the National Science Foundation (NSF) (Award No. CBET 0651739) and the National Institutes of Health (NIH) (R01GM088291). The content is solely the responsibility of the principal investigator and does not necessarily represent the official views of the NSF or NIH.

Research News & Publications Office
Georgia Institute of Technology
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Media Relations Contacts: Abby Robinson (abby@innovate.gatech.edu; 404-385-3364) or John Toon (jtoon@gatech.edu; 404-894-6986)

Writer: Abby Robinson