generation of regionally specified dopaminergic neural cells … · on a sequence of slides, and...
TRANSCRIPT
Differentiation of iPS cells into dopaminergic
neurons
Transplantation into the Rat Brain on Day
20
Inject Immunosurpressor, Cyclosporin A
Euthanization
Slicing of Serial
Coronal Sections
DAB Staining
Parkinson's disease is a neurodegenerative disease involving the malfunctioning and death of dopaminergic (DA) neurons. Due to the depletion of the neurons producing dopamine, there is a lack of t h e n e u r o t r a n s m i t t e r s r e s p o n s i b l e f o r communicating the signals necessary for movement and coordination. We found that differentiating induced pluripotent stem cells (iPSC) to DA neuron progenitors followed by transplantation into the rat brain may enable differentiation into DA neurons. After 3-4 months, we will extract the brain, mount it on a sequence of slides, and observe their differentiation. We have yet to produce consistent results. However, we will continue to repeat experimentation and review mechanics of the protocol.
Project Goals • Ensure survival of human iPS-derived neurons in
rat brain • Construct a more efficient differentiation method • Decrease the symptoms of Parkinson’s disease
with the availability of human midbrain DA neurons
• Translate this research to clinical studies
The Houbo Method Results
Discussion
Photo Credit: Houbo Jiang, Ph.D.
References
Contact information
• Utilize The Houbo Method
• Select high purity of midbrain floorplate progenitors
• Inconsistent • iPS-derived DA
neurons in 6-ODHA Rat models to produce motor impairments
• Xi, Jiajie, Yan Liu, Huisheng Liu, Hong Chen, Marina E. Emborg, and Su-Chun Zhang. "Specification of Midbrain Dopamine Neurons from Primate Pluripotent Stem Cells." Stem Cells 30.8 (2012): 1655-663.
• Kirkeby, Agnete, Shane Grealish, Daniel A. Wolf, Jenny Nelander, James Wood, Martin Lundblad, Olle Lindvall, and Malin Parmar. "Generation of Regionally Specified Neural Progenitors and Functional Neurons from Human Embryonic Stem Cells under Defined Conditions." Cell Reports 1.6 (2012): 703-14.
• Jiang, Houbo, Zhimin Xu, Ping Zhong, Yong Ren, Sonia George, Gaoyang Liang, Haley A. Schiling, Zihua Hu, Yi Zhang, Xiaomin Wang, Shengdi Chen, Patrik Brundin, Zhen Yan, and Jian Feng. "Cell Cycle and P53 Gate the Direct Conversion of Human Fibroblasts to Dopaminergic Neurons." 2015. [submitted for publication].
• "Parkinson's Disease Symptoms." The Michael J. Fox Foundation for Parkinson's Research. The Michael J. Fox Foundation, n.d. Web. 25 June 2015.
• Yamanaka, Shinya. "A Fresh Look at IPS Cells." Cell 137.1 (2009): 13-17. Web.
• "Who Has Parkinson's." Statistics on Parkinson's. Parkinson's Disease Foundation, 2015. Web. 28 June 2015.
Christina Aponte CSTEP Research Intern
Emaill: [email protected] www.linkedin.com/christinaaponte
• 1 million Americans diagnosed with Parkinson’s disease – 60,000 new patients annually
• Currently no Diagnostic Test available • Problem: Loss of Dopamine Neurons
Ø Tremors Ø REM Sleep Disorder Ø Cognitive Impairment
Figure 2: Creating iPS Cells. Usage of iPS cells generates patient-specific human midbrain dopaminergic neurons to study Parkinson’s disease.
Acknowledgments • Houbo Jiang, PhD., Research Mentor • Jian Feng, PhD., Principal Investigator • Michael J. Fox Foundation • The Collegiate Science and Technology Entry Program
Background Information
Figure 1: The Human Brain. A patient with Parkinson’s disease experiences loss of dopaminergic neurons in the substantia nigra.
Figure 3: Surgical insertion is made into the striatum of a rat brain.
Figure 6: Neural Differentiation. After 3-4 months, staining provides a visual of the differentiation that had occurred at the injection site.
Culture of induced
pluripotent stem cells
Day 1
Differentiation into
dopaminergic neurons
Day 8
Ready for Insertion
Day 20
Part 1: Differentiation of iPS Cells to DA Neurons
Part 2: Transplantation into Brain
Conclusion
dcAMP
Can produce more physiological
responses than cAMP
Is permeable
Assists in neurite growth
Prevents inactivation of second
messengers
Experimental Methods
Correct Cell
Form
Purmorphamine • Influences growth &
development of iPS cells in vitro
Ascorbic Acid • Provides
acidic environment
Heparin • Inhibits the degradation
of growth factors
Accutase • Detaches the
iPS cells from the bottom of the well
Figure 4: Floorplate Neuroepithelial Cell Formation. Arrests Cell cycle, p53 knockdown, and extracellular environment to influence neural maturation
Figure 5: DA Differentiation. Facilitates attachment, cell growth, promotion of neural morphology, motility of cells
33%
67%
Zhang Method
Fail Success
67%
33%
Parmar Method
Chart 1: Results seen in Rat brain. Other methods to culture the iPS cells in the first stage of the experiment. Failed attempts are slides that show no neural differentiation. Solution: Insert
dopaminergic neurons into the
portion of the brain experiencing
degenerative effects
Striatum
Generation of Regionally Specified Dopaminergic Neural Cells from Induced Pluripotent Stem Cells
Christina Aponte, Houbo Jiang, Ph.D., Jian Feng, Ph.D. Department of Physiology and Biophysics, The State University of New York at Buffalo
Introduction Abstract