Scientists still have a long way to go before they can harness the immense potential of human stem cells. This week in Nature journals, three separate reports lay out some of the current problems and offer some new methodologies that might move the process along.
Researchers working on neurodegeneration are seeking ways to coax stem cells to produce functional neurons, and they have strides toward this goal (ARF related news story, ARF news story, and ARF news story). And yet, in the February 13 Nature Neuroscience online, a team led by Lars Olsen at the Karolinska Institute in Stockholm, with collaborators elsewhere, cautions that neuronal stem cell grafts may cause considerable pain as a side effect.
First author Christoph Hofstetter and colleagues found that while transplanted stem cells indeed help heal spinal cord injuries in rats, it can also lead to allodynia, a skin hypersensitivity that often accompanies bouts of migraine headaches. Allodynia manifests itself as a painful reaction to normally innocuous stimuli on the skin, such as a light touch. When Hofstetter and colleagues implanted stem cells into rats that had lesions of the thoracic part of their spinal cords, they found that the animals developed allodynia of the forepaws—they quickly withdrew their paws when stimulated mechanically or in response to cold or heat.
The same stem cell transplantation did improve recovery, as the treated rats’ locomotor function was significantly better than that of untreated animals. However, the reason for this is uncertain, as the authors were unable to detect any new axonal sprouting on the distal side of the injury. They did, however, find considerable and aberrant axonal sprouting on the proximal side, and this excessive sprouting correlated with the allodynia.
Such complications could plague experimental stem cell therapies. In this case, though, the authors were able to reduce the side effect by first engineering the cells to express neurogenin-2, a transcription factor that influences cell fate and can suppress astrocyte differentiation (see ARF related news story). When Hofstetter examined grafts three weeks after implanting neurogenin-2-stem cells, he found that astrocytes only made up about three percent of the newly formed cells as opposed to about 75 percent in animals treated with normal neural stem cells. Cells expressing the neuronal marker NeuN, on the other hand, made up about 37 percent of cells, as opposed to four percent when normal cells were used. What’s more, animals given neurogenin-treated cells made statistically better behavioral recoveries, and functional MRI measurements suggested that their hind limb responses were partially restored, too. The authors suggest that “controlled differentiation” may be a way to avoid serious side effects that accompany the use of stem cell grafts.
The age-old hope for a mystic fountain of youth has inspired artists and enriched snake oil salesmen, but no one yet has truly managed to tap this legend for the benefit of aging bodies. If, indeed, there is an elixir that could rejuvenate the old among us, scientists appear to be on to some of its ingredients. In yesterday’s Nature, Irving Weissman, Thomas Rando, and colleagues at Stanford University describe an unusual experiment suggesting that substances in the blood of young mice dramatically help tissue in older animals regenerate.
The study stems from two earlier observations. In the late 1980s, John Faulkner and colleagues at the University of Michigan had shown that old muscle regenerates better when placed in young animals (see Carlson and Faulkner, 1989). More recently, Rando and colleagues found that forcing activation of the Notch pathway boosts the fitness of older muscle progenitor cells. Putting two and two together, Rando and colleagues wondered if the circulation of young animals might contain an ingredient that activates this pathway and stimulates regeneration.
To test the theory, joint first authors Irina and Michael Conboy made parabiotic mouse pairs, essentially hooking the circulatory systems of two animals together. They then tested the animals for their ability to fire up their stem cells and regenerate muscle tissue in vivo. The authors scored mice based on expression of embryonic myosin heavy chain (eMHC), a marker of regenerating muscle. Young mice (two to three months old) in parabiotic pairs got scores of around 20; old mice (19-26 months old) in pairs did worse, scoring 5 or less. But when an old mouse was plumbed up with a young mouse, both animals scored around 20—the regenerative capacity of the old matched that of the young.
Wait a second, you might say. Could the energized myogenesis in old mice be due to infiltration of stem cells from their young partners? The authors ruled this out by pairing mice that systemically express green fluorescent protein with mice that didn’t. Instead, they write that “the impaired regenerative potential of aged satellite cells can be improved by a modification of the systemic environment, by means of either an increase of positive factors in young mouse serum, or a decrease or dilution of inhibitory factors present in old mouse serum, or both.” Initial data appear to make the first possibility more likely. Conboy and colleagues detected increases in Notch signaling in older mice that were in an old/young parabiotic pair; expression of the Notch-activated protein Delta was elevated eightfold in these animals. This is not by itself an indication of positive factors, but the authors also found that Delta was elevated in skeletal muscle stem, or satellite, cells when they were exposed to serum from young mice. What’s more, Conboy and colleagues found that proliferation of liver cells increased about threefold in old mice exposed to young blood, and that this correlated with a decrease in expression of the protein brahma (Brm), which has previously been shown to reduce proliferation of hepatic progenitor cells.
The authors suggest that “it will be of great interest to identify the factors that have such a critical influence on tissue-specific progenitor cells.”
No Mouse with My Stem Cells, Please
Finally: What’s worse than a fly in your soup? Answer: Mouse molecules in your stem cells. Ever since human embryonic stem cells have been propagated, they have customarily been grown on mouse feeder cells, a fact that precludes their use in the clinic. What is badly needed, therefore, is a way to harvest and propagate human stem cells without contamination by non-human tissue.
Yesterday, in an advance online publication in Nature Methods, James Thomson and colleagues at the WiCell Research Institute and the University of Wisconsin, both in Madison, reported that the presence of two specific proteins, noggin and basic fibroblast growth factor (bFGF), persuade human stem cells to proliferate indefinitely in the absence of feeder cells.
Martin Pera, a stem cell researcher from the Australian Stem Cell Center at Monash University, writes in an accompanying news and views that this brings us “one step closer to the goal of a fully defined system for human ES cell (hESC) culture free of animal products.” One step closer because, even with noggin and bFGF, serum must still be added to keep the cells from differentiating.
This work also underscores fundamental differences between mouse and human cells. In mice, bone morphogenetic proteins (BMPs) —powerful inducers of bone cells from hematopoietic precursors—cooperate with leukemia inhibitory factor to suppress differentiation of stem cells and keep them in a state of self-renewal. In contrast, BMPs in humans have been found to induce differentiation of stem cells.
When first author Ren-He Xu and colleagues found that conditioned medium—decanted from the soup in which mouse feeder cells are grown—contains considerably less BMP activity than unconditioned medium, which can be purchased straight from a supplier, they wondered if inhibiting this activity might be an alternative to conditioning the medium. To test this idea, they grew hESCs in the presence of noggin, an antagonist of BMP, and bFGF, which is normally added to the cell cultures. They found that not only did the cells retain their capacity for self-renewal, but importantly, they didn’t lose their capacity to develop. The results suggest that researchers may be able soon to toss mouse feeder cells into the dustbin of human stem cell history.
But, as both Xu and colleagues and Pera suggest, challenges remain. For example, even without feeder cells, hESCs must still be cultured on a complex matrix, called matrigel, and an ill-defined medium containing animal products. A second drawback is that single cells do not grow well in the medium/noggin/bFGF mixture; they need to be grown in clumps.
“It seems unlikely that any one laboratory will be able to devise and validate the ideal hESC culture system in isolation. There is a very strong case for collaboration and cooperation internationally in tackling this important challenge for the field,” writes Pera. Thankfully, getting the fly out of the soup is easier.—Tom Fagan
- Adult Corticospinal Neurogenesis—Axons Run Spinal Cord Marathon
- Small Molecules Turn Stem Cells into Neurons, Targeting GSK3β
- Smells Like Neuronal Proliferation!
- Neurogenin Suppresses Astrocyte Differentiation
- Cordle A, Landreth G. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors attenuate beta-amyloid-induced microglial inflammatory responses. J Neurosci. 2005 Jan 12;25(2):299-307. PubMed.
No Available Further Reading
- Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods. 2005 Mar;2(3):185-90. PubMed.
- Pera MF. Stem cell culture, one step at a time. Nat Methods. 2005 Mar;2(3):164-5. PubMed.
- Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature. 2005 Feb 17;433(7027):760-4. PubMed.
- Hofstetter CP, Holmström NA, Lilja JA, Schweinhardt P, Hao J, Spenger C, Wiesenfeld-Hallin Z, Kurpad SN, Frisén J, Olson L. Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome. Nat Neurosci. 2005 Mar;8(3):346-53. Epub 2005 Feb 13 PubMed.