New technologies are making deriving, maintaining, characterizing and differentiating human iPS lines easier, faster and more cost-effective—and will be explored in a new short course at SLAS2014. Starting with the basics and continuing through characterization assays to lineage-specific differentiation protocols for producing physiologically relevant cell lines, the course will provide a deep-dive into the field that's rapidly changing drug discovery.
"People are extremely curious about stem cells," says David Kahler, Ph.D., former director of Laboratory Automation and the Flow Cytometry Services Laboratory at the New York Stem Cell Foundation. "Over the past few years, at SLAS annual meetings and elsewhere, I've been asked: 'How do you work with them?' 'Where can we buy them?' 'Can we make them ourselves?' 'How do we get involved?' The short course is geared toward answering those and other questions."
Kahler; Justin Ichida, Ph.D., an assistant professor of Stem cell Biology and Regenerative Medicine at the University of Southern California; and Kelvin Lam, founder and president of Simplex Pharma Advisors and cofounder of the SLAS Stem Cells and 3D Microtissues Special Interest Group, will present an Introduction to the Derivation and Maintenance of Human Induced Pluripotent Stem Cells at SLAS2014.
The increasing availability of various stem cell platforms and technologies is paving the way for a "new 'hybrid' discipline that involves collaborations between traditional stem cell scientists and traditional high-throughput screening scientists," adds Lam. Moreover, opportunities for collaboration are surfacing "within the stem cell research community itself, because so many people are specializing in different areas."
Underlying both the desire to learn more and the need for collaboration is a "tremendous excitement," says Ichida. "With stem cells, we can unlock or greatly expand our knowledge about numerous diseases. Then, hopefully, we'll be able to develop more effective treatments because we'll be able to test them on cells that are actually affected by these conditions."
The discovery that embryonic-like stem cells could be created from mature skin cells led to a Nobel Prize for Shinya Yamanaka, M.D., Ph.D. of Kyoto University. Yamanaka's reprogramming technology for generating induced pluripotent stem cells (iPS cells), first reported in 2006, laid the groundwork for the discipline as we know it today, says Kahler. "Now we can take anybody's cells and reprogram them. And because we're using cells from an adult, we can also take that person's clinical history. That gives us a lot of metadata associated with those cells, and it's something we wouldn't have if we were working with embryonic stem cells."
That said, turning back the clock on adult cells to produce stem cells for use in drug discovery or medicine is far from simple; protocols can be costly and time-consuming—generally, a couple of months from obtaining a tissue sample, reprogramming it, selecting clones or colonies of stem cells and characterizing them to ensure quality, Kahler acknowledges. But newer technologies, which will be explored in the short course, are making deriving, maintaining, characterizing and differentiating human iPS lines easier, faster and more cost-effective. "The field is moving very rapidly, and now one of the big goals for the industry is to be able to screen compounds and drugs on physiologically relevant cells—human cardiomyocytes, for example, instead of hamster cells. That's where we're heading with stem cells, and why it's important for people to understand how to work with them."
Various platforms are used to treat human fibroblasts and turn them back into stem cells. "Viral-based platforms involve integrating DNA sections into the adult cell's DNA, thereby permanently altering the cell's genome," Kahler explains. A downside of this technique is that one of those DNA sections is c-Myc, an oncogene required for cell division. "People are concerned about using cells with an oncogene engineered into them for regenerative medicine applications, for example, and that concern led to the development of the Sendai virus (an RNA virus) platform and other techniques that use oncogenes, but don't integrate DNA sections into the cell."
Newer non-viral strategies that don't involve oncogenes are starting to become available, including the use of small molecules and modified synthetic RNAs. The latter technique, which will be among those covered in the short course, drives the expression of stem cell-inducing proteins without irreversibly altering the cells' genetic material.
Ideally, stem cell-derivation technologies would produce high yields of high quality stem cells. The reality is that in many cases, "yields are very small and there's a lot of partially differentiated cellular trash that comes along with the quality cells," Kahler says. Various strategies are used to physically separate out the "trash," including manual picking of clones, magnetic beads, and Kahler's specialty, flow cytometry and fluorescence-activated cell sorting (FACS). Kahler was instrumental in developing a method that uses FACS to streamline the stem cell-selection process.
Many labs use manual techniques to select usable stem cells from a mixed cell population containing partially reprogrammed cells, Kahler notes. "These techniques involve looking at cell cultures by eye about 20 to 30 days after starting the reprogramming process, and then manually picking out a colony of programmed cells that look good. The selected cells then are placed into a dish so you have an isolated clone, and then you manually expand and split the cells," he explains. By contrast, with FACS, a week to 10 days after cells are infected with the reprogramming material, "you dissociate them and stain them with fluorescent conjugated antibodies to specific surface markers, and then you sort them out." Prospectively isolating fully reprogrammed iPS cells "cuts two to three weeks off the programming process, and also reduces the amount of expensive cell media and manual labor required to generate stem cell lines because now you are expanding an enriched population of cells expressing a unique molecular signature that identifies them as early reprogrammed induced pluripotent stem cells."
In addition to covering strategies to derive, maintain and characterize healthy stem cell lines, the SLAS2014 short course will include a look at protocols for converting human fibroblasts into disease-affected neural cells. This requires "precise combinations of transcription factors, small molecules and growth factors," explains Ichida, who will provide specific examples of the approach. "Let's say we have a patient with Lou Gehrig's disease [amyotrophic lateral sclerosis or ALS], which is caused by selective destruction of motor neurons in the spinal cord. We can now take a skin sample from a patient with the disease and coax fibroblasts out of that skin biopsy. After a month or so, single cells start growing out of the chunk of tissue. We then insert the genes for four transcription factors and wait another month. Then we take those iPS cells and in effect guide their development back into motor neurons," he summarizes.
Will those motor neurons retain the same disease characteristics as the patient? "That's the million dollar question," Ichida acknowledges. "Nobody can really say for sure, but if they do, it will allow scientists to screen on the actual diseased human cell, rather than the right cell type but from a different animal, like a mouse, or the wrong cell type from humans." Ichida is convinced that whatever causes ALS remains embedded in the stem cell because the disease is heritable, and therefore in the DNA of affected individuals.
"The power of this stem cell technology is that it allows us to study any disease that is heritable through the genome, but we don't need to know what the genetic mutations are," Ichida explains. "For ALS, for example, we can understand mechanistically, downstream of the genes, the effects of the mutations—what is actually going wrong in these neurons—and from there, look at ways to correct them." Ichida's lab has completed the first step in the process by showing that they can make bona fide motor neurons.
"Step two, which is not yet published, is that we're now studying the neurons we've produced from patients with ALS and comparing them with neurons from control groups," says Ichida. "We're seeing a big difference in the viability of the neurons between the two groups, with most of the motor neurons that come from patients showing a tendency to rapidly degenerate. That in itself suggests that these cells are recapitulating some of the disease processes." The team also found that motor neurons developed from the patients' stem cells are, unlike control cells, also overactive and constantly fire electrical impulses—a sign that has been seen clinically.
Ichida believes his group's findings regarding stem cell induced motor neurons in ALS will be similar for many other types of neurons, as well as epithelial cells, cardiomyocytes, liver and other cells. "We're still not sure how much of the actual disease process is recapitulated in induced cells, and it may be different for different diseases. But more often than not, we will be able to learn something important about a disease by doing this."
How will gaining a better understanding of stem cell biology, reprogramming techniques and the production of physiologically relevant cell lines for disease modeling affect drug discovery? In Lam's view, this knowledge will "transform the traditional drug-discovery paradigm, which takes 15 years before you finally have a compound." When small molecules are used to differentiate iPS cells into specific cell types, whether diseased or healthy, "they're not hitting a single target. They're probably modulating various signal transduction pathways, and turning on multiple biomarkers along the way," Lam explains. "Stem cell screening is not about identifying a single drug target; it allows you to identify several targets or genes involved in key pathways, pinpointing an array of potential targets for interventions."
Stem cell screening also is changing laboratory automation approaches, Lam observes. "Traditional high-throughput screening tends to involve the use of fancy robots with lots of moving parts. For stem cell screening, big robots don't make sense," he says. "Traditional screening takes about three days, then you're done with it. But stem cell assays generally take about 28 days, so you need more of a workstation approach, with a small handheld robot and a tissue culture hood. That allows you to change media, add compounds and so on under sterile conditions, which you can't do with big robots outside the hood." In the short course, Lam will compare and contrast conventional screening and stem cell screening, discuss various high content and phenotypic screening platforms for doing the latter and explain the factors—e.g., lab space, needs, budget—that participants should take into consideration when deciding on a stem cell-related technology purchase.
The excitement surrounding iPS cells and their promise notwithstanding, "we will be emphasizing that the processes involved in deriving, maintaining and differentiating stem cell lines for drug discovery and regenerative medicine applications are complex and require significant planning and a strong multidisciplinary team."
Kahler and others are trying to develop automated techniques to identify usable stem cells "because even among skilled cell culture technicians, everyone is different," Kahler says. "You can give five technicians the same cell line to start with and you'll end up with five different cell lines. We need an automated or defined, standardized way of creating cell lines that's the same every time, so that any variability in results can be attributed to the cell line or disease line, not to the technician's way of working. There is a developing awareness in the field that stem cells are reagents and must be manufactured and characterized according to a defined set of accepted standards in order to be useful for drug discovery or clinical grade regenerative medicine applications. We intend to discuss some of the important considerations for manufacturing and characterizing high quality cell lines."
These needs are also opportunities, Kahler stresses. "My opportunity was to find a way to use FACS, which enables us to isolate reprogrammed cells at an early time point. Right now, people can get fibroblasts and turn them into a stem cell line fairly easily with a plethora of techniques. The challenges and opportunities today involve taking that stem cell and turning it into the cell they want to study as quickly, cleanly and cost-effectively as possible."
Meanwhile, it behooves everyone working in drug discovery R&D today to understand both stem cell biology and stem cell screening, Lam urges. "Stem cell biology itself is not new; it's been around for more than 50 years. What's new is marrying that knowledge to drug discovery to create a hybrid discipline built around a stem cell-differentiating screening paradigm," he explains. "Like it or not, the drug-discovery field is moving rapidly in this direction on all fronts. David Kahler, Justin Ichida and I work on stem cells in different institutions and from different angles—and this short course gives us a great opportunity to come together and share our experiences with each other and with the audience."
Introduction to the Derivation and Maintenance of Human Induced Pluripotent Stem Cells will be held on Saturday, January 18 from 8:30 am - 4:30 pm at the San Diego Convention Center.
October 28, 2013