Researcher Updates
$75K Sr. Investigator Award Update
Lorraine Iacovitti, Ph.D.
Associate Director, Farber Institute for Neurosciences
Professor, Department of Neurology
Thomas Jefferson University Medical College
Research Title: Re: Studies of midbrain dopamine specification of human ES cells
$25k Jr. Investigator Award Update
Ms. Katherine Klapperich, Ph.D
Dept of Biomedical Engineering
Boston University
Research Title: Tissue Engineering Scaffolds for Guided Differentiation of neural Progenitors from Embryonic Stem Cells
$25k Jr. Investigator Award Update
Thomas Paul Zwaka, M.D.
Department of Molecular and Cellular Biology
Center for Cell and Gene Therapy
Baylor College of Medicine
Analysis of the transition from human ES cells to primitive ectoderm to neural ectoderm
$75K Sr. Investigator Award Update
Lorraine Iacovitti, Ph.D.
Associate Director, Farber Institute for Neurosciences
Professor, Department of Neurology
Thomas Jefferson University Medical College
Research Title: Re: Studies of midbrain dopamine specification of human ES cells
Parkinson’s Disease (PD) is a neurodegenerative disorder whose symptoms of tremor, rigidity, and bradykinesia are caused by the degeneration of dopamine (DA) neurons in the midbrain, the majority of which are lost prior to diagnosis. Although the symptoms of early stage PD can be alleviated by a variety of palliative treatments, efficacy declines and treatment-related side effects emerge with the inexorable progression of the disease. Consequently, cell replacement remains an important potential therapy, particularly at later stages of the illness. Towards this end, our lab has studied a variety of replacement cell types in animal models of PD, among which, human embryonic stem cells (hES) is one of the most promising. Conseqeuntly, we have previously shown that a fraction of hES-derived cells can indeed differentiate into DA neurons after transplantation into the brain, and these grafts are sometimes capable of reversing motor deficits in PD-lesioned rats (Iacovitti et al., 2007). It is now necessary to build on this success by increasing both the number and homogeneity of prospective DA neurons in the transplant. Our recent studies have therefore focused on strategies to identify hES cells which are destined to become DA neurons, define the lineage pathway they follow and amplify the stage which is most suitable for transplantation into the Parkinsonian brain. With the support of the Tilker Foundation, our studies over the last six months have established that the homeoprotein Lmx1a plays a critical role as a DA fate gene, specifying which hES cells will ultimately commit to a DA phenotype (Fig. 1A). Moreover, by simultaneously tracking the expression of other DA-specific markers, like the enzymes Adh1a1 and TH, it has been possible to delineate precise lineage steps in the fate restriction process as cells move from DA-specified progenitor and precursor cells to mature DA neurons (Fig. 1B). Using genetic engineering techniques, we are now hoping to further drive DA differentiation in these cells and to use these markers to select stage-specific cells to compare cell purity after transplantation into PD rats. In this way, we hope to move the field one step closer to a cell replacement therapy in PD.
These findings were presented at the 2nd Modern Drug Discovery & Development Summit in December 2006 in Philadelphia PA. They have also been submitted in abstract form for presentation at the Society for Neuroscience Meeting in San Diego, CA in November of 2007. In addition, a manuscript acknowledging Tilker Foundation support has been submitted for publication. Finally, because of this award, we were able to generate the necessary preliminary data for a new NIH grant application which was submitted on 6-5-07.
$25k Jr. Investigator Award Update
Ms. Katherine Klapperich, Ph.D
Dept of Biomedical Engineering
Boston University
Research Title: Tissue Engineering Scaffolds for Guided Differentiation of neural Progenitors from Embryonic Stem Cells
We have developed a cell culture platform that will allow rapid parallel screening of a library of three dimensional culture environments with gradients of mechanical properties along the length of the hydrogel. Neurite extension has been shown to be highly dependent on the stiffness of the culture system material and we expect neurite extension to be enhanced in the presence of mechanical gradients. In order to facilitate testing of cell response to a gradient environment, the Biomedical Materials and Microenvironments Laboratory has developed a technique for synthesizing gradient gels in microchannels. This technique will allow up to 10 gels to be tested in a single cell culture dish. The sides of the microchannels are first grafted with a butyl methacrylate solution to ensure covalent bonding of the poly-2-hydroxyl ethyl methacrylate hydrogels. Gradient poly-2-hydroxyethyl methacrylate gels are synthesized by simultaneously flowing two different prepolymer solutions into a microfluidic channel. After allowing the solutions to diffuse for one hour, the prepolymer solutions are crosslinked under ultraviolet light to create a gradient hydrogel. The modulus along the gradient is determined using nanoindentation on a Hysitron TriboIndenter with a 50 micron conospherical fluid-cell tip. A typical gel shows changes of 300 Pa/micron. This rapid change in modulus over a small area should allow cells to sense the direction of the gradient. Initial in vitro studies in this new system using human neural progenitor cells are underway.
$25k Jr. Investigator Award Update
Thomas Paul Zwaka, M.D.
Department of Molecular and Cellular Biology
Center for Cell and Gene Therapy
Baylor College of Medicine
In the last six months, we have focused on two areas: (1) optimization of the neuronal differentiation protocol, and (2) testing the genetically engineered human embryonic stem cell line (Tyrosine hydroxylase knock-in).
Optimization of the neuronal differentiation protocol: We previously demonstrated efficient in vitro differentiation of embryonic stem cells into neural cells in the absence of serum or after the addition of specific growth factors in multicellular aggregates (embryoid bodies). These methods consistently produce a population of cells that is over 95% positive for nestin, a marker of neural precursor cells. Preliminary results obtained in our laboratory show that this neural differentiation can be extended to dopaminergic neurons. In future, we will use knock-in cell lines to examine differentiation into dopamine-producing neurons. Our early experiments focused on establishing the basic kinetics of a solid embryoid body differentiation scheme with human embryonic stem cells.
We have established a robust and simple protocol that allows effective differentiation of human embryonic stem cells into neurons. The procedure involves a short suspension cell culture step (Fig. top left), outgrowth on a human extracellular matrix of fibronectin (Fig. top right), formation and isolation of neural rosettes (Fig., bottom left), and the final neural differentiation step (Fig., bottom, right). The human embryonic stem cell line, H9, is shown.
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