New technologies improve treatment strategies for diseases such as Parkinson’s

On March 18, Cell Stem Cell published online in the form of a cover article by the team of Chen Yuejun, a researcher at the Center for Excellence in Brain Science and Intelligent Technology (Institute of Neuroscience), Chinese Academy of Sciences, by establishing a new high-throughput lineage tracing technology across differentiation stages – SISBAR, the researchers analyzed the single-cell lineage of human pluripotent stem cells differentiated into ventral mid- and posterior brain nerve cells, and discovered many new lineage differentiation pathways and molecular regulatory mechanisms. The study demonstrates the application of new findings based on SISBAR technology in improving cell therapy strategies for Parkinson’s disease.

Dopaminergic neurons transplanted in the mouse brain Photo courtesy of interviewee

A fundamental problem in developmental and stem cell biology is how to resolve the developmental lineage between different cell types in organisms. Analyzing these lineages can provide a deeper understanding of the normal development process of living organisms and the molecular mechanisms in the pathological state, providing clues for manipulating in somatic cell differentiation, optimizing cell differentiation methods in vitro, and promoting the development of regenerative medicine based on cell replacement therapy. However, classical methods are mainly used to resolve lineage relationships between different cell types at the same differentiation/developmental time point, and cannot simultaneously provide a stage of precursor cell identity features that are related to them. How to delineate a more complete cell development pathway is an urgent problem to be solved in the field of development.

“In response to the above problems, we have developed SISBAR technology by combining virus-mediated cell barcode labeling technology, single-cell sequencing technology and clone isolation strategy.” “SISBAR technology can trace lineage-derived lineage clones of a single precursor cell across differentiation stages, while obtaining single-cell transcriptome information of the precursor cell. ”

The researchers applied SISBAR technology to the in vitro differentiation system of the human ventral mid-hindbrain, and established a lineage analysis method of “potential perspective” and “origin perspective” to analyze the differentiation potential and differentiation origin of different cell types defined by the transcriptome, so as to construct a multi-level lineage tree to describe the entire differentiation process.

“This multi-level lineage tree reveals many unreported divergent and convergent cross-stage lineage differentiation pathways during mid-hindbrain cell differentiation.” “The study also revealed the relationship between population-level lineages and clonal-level lineages across differentiation stages, and found that the fate of individual precursor cells in the same type of precursor cells (single-cell transcriptome-defined cell clusters) can be different in divergent lineage relationships; The set of differentiated fates of different precursor cells represents the differentiation fate of that precursor cell type at the population level. In converging lineages, it was found that individual cells in the same type of daughter cells can have different lineage origins, and these different origin daughter cells will carry unique genetic imprints of their parent cells. ”

The research team further demonstrated the application of SISBAR technology in the analysis of cross-differentiation stage lineage relationships and related molecular regulatory mechanisms in cell therapy for neurological diseases. Using the SISBAR technique, they found that midbrain dopaminergic neuroprecursor cells have at least three fate differentiation potentials, including midbrain dopaminergic neurons, midbrain glutamatergic neurons, and vascular pia mater-like cells.

After the researchers identified specific surface molecular markers of early midbrain dopaminergic neuroprecursor cells and transplanted them into the striatum of Parkinson’s disease model mice, the proportion of target cells-midbrain dopaminergic neurons in the graft was significantly improved. And, consistent with the results of the multi-differentiation potential of midbrain dopaminergic neural precursor cells discovered by SISBAR technology, this demonstrates the application of SISBAR technology in improving cell therapy strategies and predicting the differentiation fate of transplanted cells in vivo.

Cell replacement therapy based on human pluripotent stem cells has broad application prospects in the treatment of many diseases that are difficult to cure with traditional medicines, such as Parkinson’s disease. However, there is significant heterogeneity in donor cells (cell drugs) derived from human pluripotent stem cells, which will lead to a low proportion of target cells and instability in cell composition in donor cells and grafts, hindering the wider application of cell replacement therapy in the clinic.

It is foreseeable that as a general-purpose technology, SISBAR can be widely used in vitro differentiation systems based on human pluripotent stem cells, including neural differentiation, to construct a lineage development tree for the entire differentiation process, and to analyze the molecular regulatory mechanism of lineage differentiation, so that we can better understand the mechanism of target cells and non-target cells in the differentiation process, so as to improve cell therapy strategies and obtain safer and stable cell therapy results. At the same time, SISBAR also provides new methods and perspectives for analyzing lineage relationships during cell differentiation/development, which is an important complement to classical lineage tracing methods. (Source: Zhang Shuanghu, China Science News)

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