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.
Researchers are beginning to unlock the secrets of pluripotent stem cells, and the ability to differentiate those cells into different kinds of mature cells has exciting therapeutic value. Early applications in Parkinson’s disease and diabetes research are promising, but the complex rules governing the development of a stem cell into a mature cell are far from understood. Here’s a look at new research tools for unraveling stem cell differentiation, with a focus on therapeutic applications.
Tools and reagents
Many vendors offer tools for differentiating stem cells. Most commonly, induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are used for experimental research. Thermo Fisher Scientific’s offerings include support for differentiated stem cells such as neurons, cardiomyocytes, definitive endoderm, osteocytes and adipocytes. In December 2015, Thermo will release a new kit for differentiating iPSCs into midbrain dopaminergic neurons, which are important in Parkinson’s disease research. With this kit, “researchers can bank the precursor cells, which enables them to pause lengthy protocols, validate and adjust the differentiation process for different cell lines and share banked cells with peers,” says Alex Hannay, senior product manager in cell biology in Thermo Fisher Scientific‘s life sciences solutions group.
MilliporeSigma also offers a range of tools, including the Human ES/iPS Neurogenesis Kit, which includes all media and reagents for producing expandable neural progenitor cells that become terminally differentiated neurons. The company also offers kits to differentiate human ESCs or iPSCs to dopaminergic neurons or oligodendrocytes. The latter is especially challenging, says Vi Chu, manager of research and development at MilliporeSigma, so the Human Oligodendrocyte Kit “contains ready-to-use human oligodendrocyte progenitor cells along with optimized media for their limited expansion and differentiation into mature oligodendrocytes.” MilliporeSigma also offers kits for differentiating mesenchymal stem cells into adipocytes and osteocytes.
Bio-Techne’s range of kits enables the differentiation of stem cells into cardiomyocytes, ectoderm, definitive endoderm and mesoderm, as well as hepatocytes, with its new StemXVivo® Hepatocyte Differentiation Kit. The company also offers reagents to support differentiation of mesenchymal stem cells into chondrocytes, adipocytes or osteocytes. An advantage of kits is their standardized reagents and protocols, which often lead to better consistency. “Inconsistent differentiation efficiency due to cell-line variability is a big challenge for many laboratories,” says Joy Aho, manager of stem cell research and development at Bio-Techne. “There can also be experiment-to-experiment variability with the same cell line.”
PeproTech offers antibodies for pluripotent stem cells, mesenchymal stem cells and hemopoietic stem cells, as well as a library of proteins and small molecules, some of which are used in stem cell research. Rick Cohen, director of the Stem Cell Research Center at Rutgers University and an independent stem cell consultant for PeproTech, is helping that company optimize stem cell differentiation kits. Kits for cardiomyocytes and dopaminergic neurons, as well as media for mesenchymal stem cells, will be released in 2016.
Figuring out what’s what
Many differentiation kits include an antibody specific to a marker on the mature cell, both to verify differentiation and to estimate the extent of differentiation. A functionally mature cell—like a cardiomyocyte that beats and pulses electrically—is much easier to identify, but prior to maturity, identifiable markers are often expressed transiently. One of the most difficult things about measuring differentiation is that it’s always in flux. “The state of cell differentiation changes continuously as the cell becomes functionally mature,” says Ivan Rich, founder, chairman and CEO of HemoGenix, which has developed differentiation assays that use colony counting and flow cytometry.
Monitoring cells during the transition from the pluripotent to differentiated state is important, because some differentiation protocols yield many immature cells, says Sarah Eminli-Meissner, director of research and development at Stemgent. Indeed, one of the biggest challenges “is getting a mature and pure population of cells that do not contain any remaining pluripotent cells.” Stemgent’s StainAlive™ antibodies, which include markers for pluripotency and differentiation, are designed to let researchers monitor the differentiation process quickly and easily in live cultures. The fluorescently tagged primary antibodies bind directly to surface epitopes, thereby eliminating the need to fix the cells to monitor the status of the culture.
Despite the array of antibody tools, determining which cells become differentiated remains tricky. “There are challenges in how to monitor each stage of lineage differentiation and how to discern the different states of the resulting cells,” says Chu. MilliporeSigma’s neural differentiation kits include antibodies for differentiated cells, whereas mesenchymal cell kits include both antibodies and dyes that are selective for specific lineages.
Similarly, all Bio-Techne differentiation kits include a verification antibody for the differentiated cell type. In addition to using marker expression and morphology for identification, some cells can be identified functionally. “Cells generated using our kits have been characterized further using functional assays, such as albumin secretion by hepatocytes, as detected using our ELISA assay,” says Aho.
A new fluorescent probe from Goryo Chemical, the Kyoto Probe 1 (KP-1), is designed to distinguish human iPSCs and ESCs from differentiated cells via live-cell imaging or flow cytometry. In iPSCs and ESCs the probe is localized to mitochondria, but transporters remove the probe from cells upon differentiation, according to Kenji Kikushima, chief application specialist at Goryo Chemical .
Applications of differentiated stem cells
Vendors are offering an increasing number of tools to help stem cell researchers make the foray into clinical applications. Hannay says neural lineage cells are the most common differentiated targets he sees, thanks to the high interest—and funding—in neurodegenerative disease research. Cardiomyocytes and hepatocytes are the next most common, possibly because of their use in drug development. Thermo Fisher Scientific’s serum-free CTS™ products are designed to support clinical and translational research with “enhanced documentation, which ease[s] the burden on quality systems, simplif[ies] the regulatory filing process and reduce[s] risk throughout,” says Hannay.
Stemgent, which creates iPSCs using RNAs to reprogram human somatic cells, has recently begun developing RNA-based differentiation and transdifferentiation tools. “Stemgent is in the process of applying RNA to differentiation protocols to generate differentiated cell types in a clinically compliant manner,” says Eminli-Meissner. “This will, of course, require us to generate GMP-grade materials and protocols.”
Likewise, PeproTech is investing in building a GMP-based facility dedicated to manufacturing animal-free reagents. “They recognize that cell-based therapy is going to happen,” says Cohen, who is working on turning PeproTech’s animal-containing products into xeno-free products. “They’re trying to give clinical and translational researchers the tools to move research right into clinical applications—not only with biological compatibility, but also with regulatory issues.”
To use stem cells therapeutically, Hemogenix developed the HALO® and MSCGlo™ assays, which quantitatively measure stem cell quality and potency. To be useful, most clinical and diagnostic assays must have specific, predetermined acceptance and rejection levels; stem cells are no exception. HemoGenix’s assays help determine these levels for the use of stem cells in transplantation centers and in regenerative medicine. Stem cells with high quality and potency are likely to engraft into a patient, but “if the potency and quality are below a certain level, then you might as well throw those cells away, because they’re not going to engraft in a patient,” says Rich.
Clinical studies of differentiated stem cells have already begun—using dopaminergic neurons to treat Parkinson’s disease, for example—and more are on the horizon. As researchers learn more about the subtle complexities of differentiation, it is likely that differentiated stem cells will play an even more important role in treating today’s untreatable diseases.