Objective/Rationale:
Although using human Embryonic Stem Cells (hESCs) as a clinical therapy for Parkinson’s disease is extremely promising, there exists a number of technical obstacles. In particular, the directed differentiation of hESCs results in a heterogeneous population of both differentiated and undifferentiated (and potentially tumorigenic) stem cells. To date, there is no "winning" strategy that can directly sort this heterogeneous population for future expansion in vitro. We propose to use a label-free and highly-sensitive method of protein-functionalized microfluidic pores to sort, and subsequently recover for further expansion, hESCs that have been directed toward differentiation into dopaminergic neurons.
Project Description:
Our label-free method for characterizing cells is based on measuring a current pulse generated when a single cell passes through a microfluidic-based pore. Pulse magnitude corresponds to cell size; pulse width, to the cell’s transit time across the pore; and pulse shape to cellular shape. When the pore is functionalized with specific proteins, specific interactions between a cell-surface marker and the functionalized proteins retard the cell, thus leading to an increased pulse duration that indicates the presence of that specific biomarker.
We intend to use a "decision tree" of multiple pores, each linked sequentially to one another and each functionalized with a different protein corresponding to a different cellsurface marker, to sort hESCs that have been directed toward differentiation into dopaminergic (DA) neurons. We will use our pores to remove undifferentiated, and potentially oncogenic, stem cells from a heterogeneous hESC population by screening for embryonic antigens that are downregulated upon differentiation. Further, we will identify and sort mature DA neurons based on the expression of Dopamine Transporter (DAT) and D2 receptor.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Our project can impact Parkinson’s disease by developing a method to 1) selectively remove potentially tumorigenic undifferentiated stem cells; and 2) ultimately derive homogeneous populations of hESC and their differentiated progeny that could then be used therapeutically.
Anticipated Outcome:
Our method of sorting and characterizing a few (or single) cells expressing a particular cell-surface marker would provide a much-needed alternative for: 1) understanding hESC biology; and 2) deriving homogeneous populations of hESC and their differentiated progeny that could then be used therapeutically.
Final Outcome
We have created a new method of screening and sorting human embryonic stem cells (hESCs) that is both label- and electronics-free. Our method involves binding cells with specific cell-surface markers to specific antibodies that are functionalized within a microfluidic channel. We have demonstrated the removal of potentially oncogenic cells within a heterogeneous hESC population using our method. Further, we have demonstrated the collection of NCAM+ cells from a heterogenous population. Thus, we can use our technique to both negatively- or positively-select cells for stem-cell purification.