Zeng Lab

Overview

The Zeng laboratory studies dopaminergic differentiation of human embryonic stem cells, with an emphasis on developing methods for isolating and enriching relatively purified populations of dopaminergic neurons from those cells for transplantation therapy of Parkinson’s disease.

Characterization of Human Embryonic Stem Cells

Human embryonic stem cells (hESC), derived from the inner cell mass of preimplantation embryos, can proliferate indefinitely in culture and have the ability to differentiate into any somatic cell type both in vivo and in vitro (Thomson et al. 1998). These unique properties of hESC make them potentially useful for both in vivo cell replacement therapy and in vitro developmental studies.

Fig. 1 shows a schematic diagram of deriving and culturing hESC. The possible therapeutic use of hESC—for example, to generate dopaminergic neurons for the treatment of the neurodegenerative disorder Parkinson’s disease (PD)—has attracted great attention since the first derivation of hESC in 1998.

Before hESC can be used for treating neurodegenerative disorders, however, reliable methods to characterize hESC—in particular, to induce differentiation of hESC to specific mature cell types—must be developed. We have recently characterized three hESC lines from the NIH registry, BG01, BG02, and BG03, in great detail, and have shown that they are similar to other hESC lines in morphology, karyotype stability, expression of undifferentiated markers, and ability to differentiate into cell types of the three germ layers (Zeng et al. 2004a; Brimble et al. 2004). Detailed characterization of hESC will ultimately provide the foundation for generating cell types of clinical utility, and will therefore accelerate the realization of the therapeutic promise of pluripotent hESC.

Generation of dopaminergic neurons from hESC for transplantation therapy

Dopaminergic neurons play an important role in modulating motor control, and the selective degeneration of dopaminergic neurons in substantia nigra is a hallmark of PD. Restorative therapy involving transplantation of dopaminergic neurons into the brain is one of the most promising strategies for treatment of PD, but the availability of human dopaminergic neurons for this purpose is limited because so far those neurons can be obtained only from human fetal tissues or early postnatal donors. The recent derivation of hESC, however, might provide an unlimited source of dopaminergic neurons for in vivo transplantation studies using an animal PD model.

We and others have shown that midbrain tyrosine hydroxylase (TH)-positive dopaminergic neurons can be readily generated from hESC by co-culturing with mouse stromal cell lines, or via embryoid body formation followed by selection of neural precursors (Perrier et al. 2004; Zeng et al. 2004b; Yang et al. 2005). An example of TH-positive dopaminergic neurons generated from hESC is illustrated.

fig. 2. The major limitation of these methods of generating dopaminergic neurons from hESC, however, is the low yield of TH-positive cells and the presence of other undefined neuronal and nonneuronal cell types in the heterogeneous differentiating hESC population. One approach to obtaining a purified cell population is through cell sorting, using markers expressed on the surface of a particular type of cells. We are currently trying to develop assays and markers that permit accurate and reliable characterization of the state during dopaminergic differentiation of hESC, and we have tested several neuronal surface markers (e.g., NCAM) for enriching dopaminergic precursors or neurons derived from hESC, using fluorescence-activated cell sorting (FACS). We will investigate the long-term survival and integration into the host brain of FACS-purified hESC after transplantation in a rat PD model, and evaluate behavioral recovery in animals transplanted with FACS-purified hESC. The long-term goal is to define and purify a cell population optimal for transplantation therapy of PD.

The roles of transcription factors in dopaminergic development of hESC

Precisely timed expression of transcription factors and coordinated interaction of transcription factors with other transcription factors and cell signaling molecules are thought to involve normal gene expression during development, including dopaminergic development in the midbrain. These may include not only mechanisms to direct dopaminergic neuronal differentiation and to maintain dopaminergic phenotype but also mechanisms to promote survival of dopaminergic neurons. Two transcription factors, Nurr1 and Pitx3, in particular, have been implied in both maintaining a dopaminergic phenotype and promoting survival of dopaminergic neurons. Understanding how Nurr1 and Pitx3 influence these processes may lead to therapeutic insights relevant to PD, and may be important for modifying hESC in order to generate an optimal cell source for transplantation therapy of PD.

One approach to addressing the role of Nurr1 and Pitx3 in human dopaminergic development is through gain-of-function analysis via genetic modification or protein delivery. Our hypothesis is that Nurr1 and Pitx3 may regulate, independently or cooperatively, dopaminergic development and maturation in hESC, and that their roles can be assessed through gain-of-function analysis. We have constructed Nurr1- and Pitx3-containing vectors for overexpression in mammalian cells and are currently testing the effects of these transcription factors on dopaminergic differentiation in hESC.

References

Brimble, S., Zeng, X., Weiler, D. A., Luo, Y., Liu, Y., Lyons, I. G., Freed, W. J., Rao, M. S., Robins, A. J., and Schulz, T. C. 2004. Karyotypic stability, genotyping, differentiation, feeder free maintenance and gene expression sampling in three human embryonic stem cell lines derived prior to August 9th 2001. Stem Cells and Development 13(6):585–597.

Perrier, A. L., Tabar, V., Barberi, T., Rubio, M. E., Bruses, J., Topf, N., Harrison, N. L., Studer, L. 2004. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 101(34):12543–12548.

Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282(5391):1145–1147.

Yan, Y., Yang, D., Zarnowska, E. D., Du, Z., Werbel, B., Valliere, C., Pearce, R. A., Thomson, J. A., Zhang, S. C. 2005. Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 23(6):781–790.

Zeng, X., Cai, J., Chen, J., Luo, Y., You, Z. B., Fotter, E., Wang, Y., Harvey, B., Miura, T., Backman, C., Chen, G. J., Rao, M. S., Freed, W. J. 2004b. Dopaminergic differentiation of human embryonic stem cells. Stem Cells 22(6):925–940.

Zeng, X., Miura, T., Luo, Y., Bhattacharya, B., Condie, B., Chen, J., Ginis, I., Lyons, I., Mejido, J., Puri, R. K., Rao, M. S., and Freed, W. J. 2004a. Properties of pluripotent human embryonic stem cells BG01 and BG02. Stem Cells 22:292–312.