Stem cell therapy is a new paradigm of translational medicine and regenerative medicine. Researchers and clinicians strive to develop the unique potential of stem cells for future treatment of diseases characterized by tissue loss and degradation. Photogenetic methods allow targeted activation or inactivation of selected cell types and individual cell functions, including stem cells, with high precision and good temporal and spatial discrimination.
Figure 1: An integrated in vitro approach of induced pluripotent stem cells (iPSCs) and optogenetics for the study of neurological disorders. (Somuncu ÖS, 2020) Creative BioMart provides the construction of stable opsin expressing stem cell lines. We have successfully constructed stable mouse embryonic stem cell lines expressing high levels of ChR2 for our customers. Photogenetics is helpful to reveal the main therapeutic mechanism of stem cell therapy from two aspects: (1) promote and improve the neuronal differentiation of stem cells expressing ChR2; (2) Improve and test the functional integration of stem cell-derived neurons and endogenous circuits after transplantation.
| Step | Service Description |
| Expression strategy design | According to your research question, select the promoter expressed in the whole differentiation process, such as elongation factor 1 α (EF1 α), or promoters active only in specific lineages. Opsin fused directly with fluorophore was selected so that the membrane binding position could be determined by fluorescence microscope. |
| AVV vector design and transformation | Due to the diversity of infection, viral transduction of (stem) cells using viral vectors will always lead to the heterogeneity of expression intensity, resulting in the insertion of multiple copies of GOI into the genome. Therefore, the sorting step of selecting only highly expressed but dynamic cells is very important. Cell viability can be assessed by propidium iodide (PI)/annexin V (anv) staining, using fluorescence activated cell sorting (FACS), ideally sorting double negative populations in the same step. Stem cells were cultured with opsin fluorophore encoding lentivirus for 24 hours. |
| Induction of neuronal differentiation | During differentiation, the expression intensity of ChR2 - for example, by ubiquitous promoters (such as EF1 α) Drive - will decline slowly, not due to gene silencing, but most likely due to the lower overall protein expression level in neurons compared with rapidly dividing stem cells. |
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