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: caaponte@buffalo.edu 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