Systems, methods, and apparatus for induced pluripotent stem cell isolation and combinatorial production
Described herein are various systems, methods, and apparatus for systematic creation of isolated homogeneous colonies of cells from vector-based lineages. The vector-based lineages may originate from multiple types of viral vector families (e.g., Paramyx-oviridae, Retroviridae, Parvoviridae) or non-natural engineered vectors or a plurality of vector combinations, for example. In certain embodiments, the isolated homogeneous colonies of cells are vector-free sub-colonies; in other embodiments, the isolated homogeneous colonies of cells are homogeneous vector sub-colonies. In other embodiments, vector mixed sub-colonies are created. The disclosed systems, methods, and apparatus are useful for inducible pluripotent stem cell (iPSC) production and work by selectively binding to one or more corresponding protein markers expressed on the surface of a cell that indicate that cellular reprogramming has occurred. Software is used to automate the purification and isolation of the iPSCs produced.
1. A method for isolating and producing induced pluripotent stem cells (iPSCs), the method comprising:
providing a colony of cells having been contacted with one or more vectors;
detecting a plurality of cell surface markers expressed on the surface of the iPSCs, wherein each of the plurality of cell surface markers is indicative of the one or more vector contacted with the colony of cells;
mapping, by a processor of a computer device based on binding specificity, a plurality of candidate binding agents to the plurality of cell surface markers;
selecting at least one of the mapped plurality of candidate binding agents for specific binding to at least one of the plurality of cell surface markers expressed on the surface of the iPSCs;
contacting the colony of cells with the at least one of the selected binding agents; and
separating the iPSCs bound to the selected binding agents from the colony of cells.
2. The method of claim 1 , wherein the colony of cells comprises somatic cells.
3. The method of claim 1 , wherein each vector of the one or more vectors comprises multiple vector families.
4. The method of claim 1 , wherein the plurality of candidate binding agents comprise one or more members selected from the group consisting of an antibody, a biotinylated bead, a hydrophobic bead, and a magnetic nanoparticle.
5. The method of claim 1 , wherein separating the iPSCs bound to the selected binding agents from the colony of cells comprises:
separating the iPSCs into vector-based lineages using one or more members selected from the group consisting of single bead separation, column chromatography, serial separation, microfluidic channel separation, magnetic bead binding, fluorescence activated cell (FACS) sorting, and antibody binding.
6. The method of claim 1 , further comprising:
determining, by the processor, a prescribed order in which the selected binding agents are to be contacted with the colony of cells for desired specific binding.
7. The method of claim 1 , further comprising:
generating one or more isolated homogeneous colonies of iPSCs from a single vector lineage.
8. The method of claim 7 , wherein the one or more isolated homogeneous colonies of iPSCs comprise a genetic variant of the colony of cells.
9. The method of claim 7 , wherein the one or more isolated homogeneous colonies of iPSCs are vector-free.
10. The method of claim 1 , wherein the plurality of cell surface markers is produced by one or more members selected from the group consisting of Paramyxoviridae virus vector family, Retroviridae virus vector family, Parvoviridae virus vector family, and a non-natural engineering vector.
11. The method of claim 10 , wherein the plurality of cell surface markers is produced by Paramyxoviridae virus vector family and wherein the Paramyxoviridae virus vector family comprises one or more members selected from the group consisting of Atlantic salmon paramyxovirus, Newcastle disease virus, Mumps virus, Ferlavirus, Hendravirus, Nipahvirus, Measels, Sendai, Avian pneumovirus, Tioman virus, and TPMV-like virus.
12. The method of claim 11 , wherein the plurality of cell surface markers comprise one or more members selected from the group consisting of Nucleocapsid protein (NP), Phosphoprotein (P), Matrix protein (M), Fusion protein (F), and Hemagglutinin-Neuraminidase (HN), Large protein (L).