Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Delivering nucleic acids into cells via transfection is a mainstay of cell and molecular biology and is being developed into therapeutic tools to combat numerous diseases. Any researchers who transfect cells must optimize conditions to find the right balance between the sometimes lethal effects of transfecting and a level of transfection efficiency that is workable and reliable. Stem cells pose additional challenges, to which there are no one-size-fits-all solutions. “Some people dream of a ‘universal’ protocol, reagent or instrument that works perfectly with all cell types, but in reality, different cell types have different transfection requirements,” says Chris Linnevers, cell biology global product manager at Bio-Rad Laboratories.
Induced pluripotent stem cells (iPSCs) are a valuable resource in both basic research and therapeutic applications—but they are more challenging to transfect than most cell lines, and not all transfection methods can be used. Here’s a rundown on technologies that are being used successfully to transfect stem cells today.
Lipid-based reagents
The well-established Lipofectamine line of lipid-based reagents from Thermo Fisher Scientific has been used to transfect many types of cells and is also effective at delivering mRNA, DNA or siRNA into stem cells. Nektaria Andronikou, staff scientist in cell biology research and development at Thermo Fisher Scientific, recommends choosing transfection tools first according to the application—such as loss of function or gain of function—and then according to cell type. “There are a variety of stem cell types, and each will require a different delivery strategy,” she says. Stem cells can vary “depending on how they were reprogrammed, the patient derivation, disease conditions or differentiated state,” says Andronikou.
Looking toward the future, Andronikou sees a need for a safe and footprint-free reprogramming method that would enable researchers to use stem cells as therapies against cancer, and for the study of monogenic and isogenic diseases. She also sees a significant need for improved methods to reprogram blood cells, because these “are much easier to harvest from patients and hold true potential of becoming a personalized therapy in the future,” she says.
If you are browsing lipid-based reagents, another source is Stemgent, whose proprietary, lipid-based transfection technology has been used to deliver RNA to iPSCs, hematopoietic and other types of stem cells. Brad Hamilton, senior director of scientific and business development at Stemgent, says that researchers are using Stemfect RNA Transfection technology “to deliver transcription factors and associated genes for the generation of iPSCs from somatic cell types” in cellular reprogramming. Stemfect also can deliver RNA to iPSCs in directed differentiation and “facilitate transdifferentiation—the c onversion from one cell lineage to another, circumventing the need to transition through the pluripotent cell state,” says Hamilton. Even though transfection technologies are bound to evolve, Hamilton believes it’s important to stay focused on RNA delivery, because “it’s the most clinically compliant way to deliver genes to convert cell types for potential therapeutic applications.”
Yet another supplier is Bio-Rad Laboratories, whose lipid-based reagent TransFectin™ has been used to transfect neural stem cells, says Linnevers. In addition, Bio-Rad offers electroporation tools for stem cell transfection (see below).
Polymer-based reagents
One of the challenges of working with stem cells is that every type is different. If you’re working with especially sensitive cells, sometimes polymer-based transfection reagents can be a gentler alternative. Laura Juckem, research and development group leader at Mirus Bio, emphasizes the importance of stem cell protocols “that allow the cells to maintain their pluripotent state” and minimize the cytotoxicity of the transfection process. Mirus Bio’s TransIT®-LT1 (Low Toxicity 1) is the company’s gentlest nonliposomal reagent, says Juckem, and can help minimize the stresses of transfection. “Stem cells, and in particular iPSCs, can be very sensitive to environmental changes and do not respond well to stress,” she says. Besides human iPSCs, Juckem says that mesenchymal stem cells also can be transfected successfully with Mirus Bio’s polymer-based TransIT®-2020 Transfection Reagent.
Electroporation
If your particular stem cells aren’t happy with lipid- or polymer-based methods, don’t despair—electroporation may work. Mirus Bio recommends combining its Ingenio® Electroporation Solution with the Nucleofector® II/2B electroporation system from Lonza for high-efficiency transfection of human iPSCs.
In fact, stem cells are particularly amenable to electroporation—Bio-Rad’s Gene Pulser Xcell™ is that company’s most widely cited transfection system for stem cell lines. One reason for this is that stem cells tend to grow in colonies, and “cells in the middle of the cluster may have less exposure to chemical or lipid-based transfection reagents, resulting in suboptimal transfection efficiency for these already difficult-to-transfect cells,” says Frank Hsiung, cell biology staff scientist at Bio-Rad. Recently, the Gene Pulser Xcell system was used to transfect targeting and guide vectors into human pluripotent stem cells to make neural lineage reporter lines, as well as “to correct SOD1 point mutations in iPSCs derived from patients with amyotrophic lateral sclerosis (ALS),” says Linnevers.
MaxCyte offers an automated electroporation platform for use on stem cells for both research and therapeutic applications. According to Madhusudan Peshwa, executive vice president of research and development at MaxCyte, cell modification can help make “stem cells more potent as a therapeutic agent.” And “in research applications, [stem cells] are being used as a more physiologically relevant tool in screening assays for small molecules.” MaxCyte can target processes in the cytoplasm or in the nucleus, depending on the types of molecules transfected.
Transfecting a molecule that affects cytoplasmic events, while not permanent, can achieve the desired effect if implemented within a physiologically functional window of time, says Peshwa. Examples include increasing stem cells’ homing activity to modify disease outcomes, or prompting cells to secrete cytokines or growth factors that synergistically help “to orchestrate an entire cascade that helps with regeneration,” he says.
Controlling cellular events at the DNA level using genome-editing tools—such as CRISPR (clustered regularly interspaced short palindromic repeat), TALEN (transcription activator-like effector nuclease) or ZFNs (zinc finger nucleases)—enables permanent changes. Even though the transfection itself is transient, the effect of the transiently expressed gene-editing tools is permanent. “You only want the [gene-editing molecules] to be present for a short duration,” says Peshwa. “But the impact of the change is … for the life of the cell.”
Recently, inquiries from scientists regarding genome-editing tools are commonly heard by vendors of transcription reagents. Thermo Fisher Scientific’s Andronikou says researchers are increasingly interested in using “stem cells for CRISPR or TALEN-based gene-editing experiments to generate disease-modeled cell types.” Mirus Bio has used its TransIT® Transfection Reagents and Ingenio Electroporation Kits with CRISPR, TALEN and ZFN strategies. “In the future, we expect to see optimized stem cell transfection protocols for CRISPR applications,” says Juckem. As stem cell transfection becomes easier and more efficient, the incorporation of other strategies—such as genome editing—is boosting the power and broadening the possibilities for today’s stem cell research.