Gene Therapy and Stem Cells Lecture

Study Notes

Hematopoietic stem cells (HSCs) are used for gene therapy in adults and children.
HSCs are readily accessible from the patient's bone marrow, reducing the risk of immune rejection upon reintroduction.
They exhibit rapid in vitro replication, longevity, and have the capacity to differentiate into both red and white blood cells.

The basic concept of iPSC is to use genes involved in cell development to push a somatic cell back to an earlier stage of pluripotency.
The first trail to make iPSC involved using retroviruses to deliver four transgenes (OCT3/4, SOX2, c-MYC, and KLF4) into fibroblasts.
Expression of these four genes, which encode transcription factors involved in cell development, “reprograms” the fibroblasts back to an earlier stage of differentiation.

c-MYC is not essential for cell reprogramming, but increases the process efficiency by increasing the proliferation rate.
KLF4 increases the p21 protein levels, decreasing the proliferation rates and p53 levels, thereby reducing apoptosis risk.
SOX-2 regulates phenotypic maintenance of stem cells at the mitosis M-G1 transition.
OCT4 represses genes related to cell differentiation, preserving cell pluripotency.

Organoids are three-dimensional (3D) cellular clusters that self-organize themselves into structures that resemble mini-organs and exhibit similar organ functionality and architecture.
Organoids can be composed from pluripotent stem cells or adult stem cells.
Organ-specific biochemical cues and morphogens (Fibroblast Growth Factor, Wnt, Sonic Hedgehog) are used to control several signaling pathways involved in the proliferation, migration, and differentiation of stem cells.

Laminin, fibronectin, collagen, and Matrigel are examples of physical supportive matrix components used in organoids.
Matrigel is a commercially available cell culture product obtained from reconstituted basement membrane extracted from Engelbreth-Holm-Swarm mouse sarcoma.
Supporting matrix is also available from decellularized matrices, biomaterial customization, and nanoparticles.

3D bioprinting aims to construct living, functional organs for reconstructive medicine by printing living cells and matrices in a 3D structure that resembles the shape of the organ to be generated.
The 3D bioprinting process comprises: obtaining a 3D image of the organ to be printed, selecting the bioink composition, and the actual printing procedure, culturing, and finally the organ quality control.

Combining cells with biomaterials can be done through top-down printing or bottom-up printing.
Top-down printing disperses cells homogeneously into biomaterials and shapes them to resemble the desired organ.
Bottom-up printing constructs the organ layer by layer, using different types of bioinks in parallel, with different types of cells, hydrogels, and soluble compounds.