Programming Human ESC-derived Neural Stem Cells with MEF2C for Transplantation in Stroke

Funding Type: 
Early Translational IV
Grant Number: 
TR4-06788-A
Investigator: 
Award Value: 
$1,020,815
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Status: 
Terminated
Public Abstract: 

The goal of this project is to produce a stem cell-based therapy for stroke (also known as an ischemic cerebral infarct). Stroke is the third leading cause of death in the USA, and a leading cause of disability among adults. Currently, there are no effective treatments once a stroke has occurred (termed completed stroke). In this proposal, we aim to develop human stem cells for therapeutic transplantation to treat stroke. Potential benefits will outweigh risks because only patients with severe strokes that have compromised activities of daily living to an extreme degree will initially be treated. Using a novel approach, we will generate stem cells that do not form tumors, but instead only make new nerve cells. We will give drugs to avoid rejection of the transplanted cells. Thus, the treatment should be safe. We will first test the cells in stroke models in rodents (mice and rats) in preparation for a human clinical trial. We will collect comprehensive data on the mice and rats to determine if the stem cells indeed become new nerve cells to replace the damaged tissue and to assess if the behavior of the mice and rats has improved. If successfully developed and commercialized, this approach has the potential for revolutionizing stroke therapy.

Statement of Benefit to California: 

The goal of this project is to produce a stem cell-based therapy for stroke (also known as an ischemic cerebral infarct). Stroke is the third leading cause of death in the State of California, and a leading cause of disability among adults. Currently, there are no effective treatments once a stroke has occurred (termed completed stroke), and the quality of life is severely compromised in those that survive the malady. In this proposal, we aim to develop human stem cells for therapeutic transplantation to treat stroke. Using a novel approach, we will generate stem cells that do not form tumors, but instead only make new nerve cells. If successfully developed and commercialized, this approach could provide a therapeutic candidate for the unmet medical need, which would have a tremendous impact on the quality of life for the patient, his or her family, and for the economic and emotional burden on the State of California and its citizens.

Progress Report: 

Stroke is the third leading cause of death in the USA and remains a great medical problem in California. Currently, there is no effective treatment for patients with a stroke who are seen several hours after the event. The goal of this project is to establish the feasibility of using a stem cell line for cell-replacement therapy to target stroke (cerebral ischemia). Using a novel, genetic pre-programming approach, we will generate human neural stem/progenitor cells (hNSC/NPCs) that are resistant to apoptotic cell death and destined to become nerve cells (or neurons). Our approach also avoids tumor formation, which can occur if stem cells that are not programmed to become neurons are injected into the brain. We will achieve our goals by introducing a constitutively active form of the transcription factor MEF2C (MEF2CA) into human embryonic stem cell (hESC)-derived hNPCs. For this purpose it is critical to identify a viral vector system that is the safest and most effective in producing MEF2CA-programmed hNPCs. We decided to use an adenoviral-associated virus (AAV) vector system because unlike other viral delivery methods (e.g., lentiviral, retroviral), an AAV system allows us to achieve MEF2CA expression in an integration-free and transient manner, as required for proper neuronal differentiation from NPCs. After in vitro characterization of these cells, including their neurogenic capacity, scalability etc., we will transplant them into rodent models of focal stroke. We are analyzing transplanted rats with immunohistochemical, electrophysiological, and behavioral methods to determine whether MEF2CA-programmed hNPCs can successfully differentiate into functional, integrated neurons into the host brain and ameliorate stroke-induced behavioral deficits. To assess the robustness of the AAV approach, we will also compare the results obtained from this system to those obtained using a hESC line that is stably programmed (resulting in permanent insertion of the MEF2C transgene into the genome of the cell, as opposed to transient MEF2C expression achieved with the AAV system). These studies will allow us to determine the effectiveness of the integration-free AAV system vs. stable integration of MEF2C on hNPCs developed for cell replacement therapy. During the current reporting period (Year 01), we have efficiently produced an AAV vector that transduces the MEF2CA transgene into hESC-derived NPCs. FACS analysis revealed that we have robustly infected the hNPCs with this AAV-based construct (~95% of cells infected). In vitro evaluation for protein and mRNA (through immunocytochemistry, and qRT-PCR assays) from the cells infected with AAV-MEF2CA revealed their neural progenitor cell identity and that the MEF2CA transgene is active in these cells. We have begun to transplant these cells into the brain of the spontaneously hypertensive (SHR) rat model of focal stroke. We have begun to compare the effects in stroke of the AAV-MEF2CA and stable-MEF2CA cell lines. In vitro characterization of the stable MEF2CA stem cell line demonstrated that we can differentiate these hNPCs into neurons. For example, protein, mRNA, and morphological analyses revealed robust differentiation of these hNPCs into mature cerebrocortical neurons. Electrophysiological analysis further confirmed the expression of functional neuronal channels and synaptic currents. Behavioral evaluation performed 12-weeks after transplant into the stroked brain with the stable-MEF2CA hNPC line vs. control revealed promising behavioral improvements in the MEF2CA-NPC transplanted group compared to control without apparent side effects.