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Living Up to Potential

Thu, 07/22/2010 - 11:24am
Jeffrey M. Perkel

Researchers are intrigued by the possibilities of Human pluripotent stem cells (hPSCs) in basic science and drug development, but much work remains if expectations are to be met.

It's a dizzying time to be studying human pluripotent stem cells (hPSCs).

Academic and industry researchers, recognizing the potential of hPSCs to basic science and drug development, and even as therapeutics in their own right, are probing these cells' biological secrets at a blistering pace. Barely does the ink dry on published data before new findings emerge to extend them. Shinya Yamanaka's seminal 2007 paper on reprogrammed human "induced pluripotent stem cells" (iPSCs),1 for instance, has been cited nearly 1,300 times to date—300 in 2010 alone, according to the ISI Web of Science.

Induced Neuronal Cells
Induced Neuronal Cells (Source: M. Wernig)

Such a dynamic and evolving research area requires an equally dynamic tool set, and developers have released a steady stream of products to enable this work. Yet unmet needs remain. Researchers can for the most part maintain stem cells in a pluripotent (that is, undifferentiated) state, but it isn't easy; hPSCs like human embryonic stem cells (hESCs) are perhaps the most high-maintenance cells in the tissue culture hood, requiring constant care and feeding. And directing these cells into specified lineages—the real promise of hPSCs—remains hit-or-miss.
"It's safe to say we have a very, very long way to go to consistently get the undifferentiated cells to form the differentiated cells we want," says Clive Glover, Senior Product Manager at STEMCELL Technologies.

Defined Growth Conditions
The promise of hPSCs lies in their pluripotency—their ability to differentiate into any cell type in the body. If researchers can define the molecular triggers that direct these cells to morph into, say, cardiomyocytes, they then would have a platform to study both how disease derails that transformation, as well as how drugs can promote or inhibit it. Eventually, they may even be able to use hPSC-derived cells as therapeutics in their own right, to replace damaged heart tissue following a heart attack, for instance.

A clinical trial based on that premise is in fact in the wings. Geron won US Food and Drug Administration approval for a Phase I clinical trial of its hESC-derived treatment for spinal cord injuries (GRNOPC1) in January 2009. Heralded as the first hESC-based therapeutic to reach human clinical trials, the study was put on hold this past August due to "microscopic cysts" at the injury site in some treated animals. According to a company statement, the trial should resume later this year following completion of a preclinical study using "new candidate markers and assays."2

For the most part, however, standard hPSC culture conditions are not particularly well suited to clinical development. These cells traditionally are maintained on a feeder layer of mouse embryonic fibroblasts (or in feeder-conditioned media) and grown in media containing animal sera, both of which supply needed, but unknown, growth factors—X-factors, basically. The potential for xenobiotic infection and batch-to-batch variability inherent in such conditions, means FDA approval of any therapeutic derived under such conditions could be slow in coming. Some popular feeder-free alternatives, such as BD Biosciences' BD Matrigel and Life Technologies' Geltrex, although better defined than MEFs, are also of animal origin.

Human iPS cells
Human iPS cells generated using Millipore’s STEMCCA constitutive polycistronic (Source: OKSM)

Recently, progress has been made in the development of fully defined, animal-free growth conditions. Both animal component-free and defined media formulations are available, and include Life Technologies' StemPro hESC SFM and KnockOut SR XenoFree, Millipore's HEScGRO, and STEMCELL Technologies' TeSR2. Xenobiotic-free growth matrices include Life Technologies' CELLstart, BD Biosciences' BD Laminin/Entactin, and Millipore's vitronectin. Or users can stick with human feeder cells, such as Millipore's forthcoming xenobiotic-free human foreskin fibroblasts. But according to Vi Chu, R&D Manager for stem cell and cell biology teams at Millipore, the market is moving in a different direction.

"The market is trending more towards chemically defined media on chemically defined substrates," Chu says.

In June three groups independently described fully defined, novel growth surface coatings that support the long-term maintenance of hPSCs: a synthetic polymer called PMEDSAH,3 a recombinant embryonic extracellular matrix protein called laminin-511,5 and a biosynthetic hybrid called a "synthetic peptide-acrylate surface" (PAS).4 And in July, three independent research teams demonstrated that human iPSCs may be generated from peripheral blood instead of via skin biopsy, an achievement that Yamanaka, in an accompanying editorial, said “represent[s] a huge and important progression in the field.”

"The fact that three groups independently came up with substances to replace Matrigel or feeder cells tells you the importance of the challenge that has been out there for years," says Joerg Lahann of the University of Michigan, a co-author on the PMEDSAH study.3

According to Lahann, such materials, combined with defined media, "will give the stem cell community a huge push forward," by enabling researchers to ask questions not otherwise possible using X-factor formulations—questions such as, what is the impact of a given growth factor, chemical, or surface on stem cell maintenance and differentiation.

Otherwise, he says, there simply are too many unknowns. "Simply [put], your experimental setup is not fully defined, which makes it very difficult to figure out what the effect of your drug candidate is," Lahann says. "It's often impossible."

Induced pluripotency and more
For all their potential, hESCs represent a painful labor of love, requiring daily care and diligent maintenance. "We have researchers that have to come in on long holidays to make sure the cells are fed and taken care of," says Chu. But for many researchers, the strike against hESCs isn't effort, but morality; some scientists simply refuse to work with cells obtained by destroying human blastocysts.

colony of hES cells growing
A colony of hES cells growing on a polymer PMEDSAH in the presence of defined cell culture media. (Source: Himabindu Nandivada, PhD, University of Michigan)

iPSCs, though, induce no such qualms.

hESC-like cells produced by introducing four DNA-binding proteins into adult cells (such as skin cells), iPSCs allow stem cell researchers to have their cake and eat it too, developmentally speaking (and assuming they faithfully reproduce hESC biology, which is still a matter of debate).

They offer other advantages, too. Most obviously, iPSCs provide a source of pluripotent progenitor cells from adults affected with genetic abnormalities, which may be used to probe the molecular missteps that bridge genotype and phenotype.

In June, researchers at the Mount Sinai School of Medicine in New York created iPSCs from two individuals with LEOPARD syndrome, an autosomal dominant disorder.6 Using these cells the authors recapitulated on a cellular level several hallmarks of the disease, such as enlarged cardiac cells, and identified potential protein players in disease biology.

Yet other research casts doubt on the promise of iPSCs, at least as they currently are produced. In February, investigators at Stem Cell and Regenerative Medicine International and at Advanced Cell Technology reported that iPSC differentiation to the hemangioblast, endothelial, and hemapoietic lineages was less efficient than that of hESCs, and that the differentiated cells expanded less productively.9 Similarly, in March Su-Chun Zhang of the University of Wisconsin, Madison, and colleagues reported that neuronal differentiation from iPSCs proceeds less efficiently than do cells differentiated from hESCs.

That difference can be problematic, says Zhang. The process of iPSC generation is already inefficient; perhaps only 1% of transduced cells will become iPSCs. If only 10% to 20% of those cells then differentiate into desired lineages, researchers will be hamstrung by a lack of material. "There's still room to improve the technology," he says.

There also is a novel alternative. In generating iPSCs, explains Marius Wernig at the Stanford University School of Medicine, researchers essentially force cells to "go back in time," dialing a cell's developmental clock back to a more premature state. Oftentimes, they then push those cells down another developmental lineage, so that what once were skin cells are now, for instance, neurons.

Wernig wanted to know whether the cells could actually make that skin-to-neuron transformation without first undergoing developmental regression.

Commercial Stem Cell Tools

Researchers interested in creating and/or differentiating hPSCs in their own lab have a wide (and ever growing) array of commercial products at their disposal.

Millipore has combined the four Yamanaka transcription factor genes in a single construct called the STEMCCA constitutive polycistronic lentivirus, with or without flanking loxP sites for transgene excision, and also offers a Human iPS Selection Kit to identify and purify reprogrammed cells.

To maintain those cells, in addition to defined and xeno-free media researchers can also obtain the three newly described surface coatings. Corning offers PAS-coated plates (jointly developed with Geron) under the brand name Synthemax. PMEDSAH-coated plates are available in limited quantities from Lahann's lab. And recombinant human laminin-511 is available from BioLamina AB, a startup company founded by Karl Tryggvason, lead author of the laminin study.4

Specialized culture dishes called AggreWell plates, available from STEMCELL Technologies, enable researchers to control embryoid body formation—a critical, but normally heterogeneous step in the stem cell differentiation process. If you want to genetically alter the cells first, consider Lonza's Amaxa Nucleofector system, which can deliver nucleic acids to fragile stem cells with minimal cytotoxicity.

For those looking to track pluripotency or differentiation, Life Technologies' TaqMan Stem Cell Pluripotency Arrays profile the expression of some 92 pluripotency-associated genes, while BD Biosciences' Human Cell Surface Marker Screening Panel enables cellular fingerprinting based on flow cytometric analysis of 242 distinct cell surface markers, thereby enabling researchers to identify molecular fingerprints of differentiation and subtle distinctions among different lines.

Finally, for those looking to circumvent manual stem cell passaging altogether, there's Cyntellect's LEAP Cell Processing Workstation and its Stem Cell Colony Purification application.

Released at the International Society for Stem Cell Research meeting in June, this application automates stem cell colony passaging and purification—normally tedious, imprecise, and contamination-prone processes—by using laser light to destroy unwanted cells while protecting fragile hPSCs from unnecessary tinkering, according to Fred Koller, Cyntellect's Chief Technology Officer.

"Most other ways to [process stem cells] involve manipulating the cells you want to keep," Koller says. "We envisioned a technology that leaves the cells you want to keep alone and instead causes problems for the cells you don't."

Mirroring Yamanaka's approach to developing iPSCs, Wernig and his team identified three transcription factors that when introduced into adult mouse fibroblasts, turn them into functioning neurons.8 The process was both rapid (initiating within three days) and efficient, with up to 20% of cells converting to neurons.

"These cells not only look like neurons and express markers associated with neurons, but these cells also behave like neurons," says Wernig; for instance, they can fire action potentials, form synaptic contacts, and "talk" to other neurons. Wernig calls these cells "induced neuronal," or iN cells.

Wernig speculates that it should theoretically be possible to use this approach to produce any cell type in the body, assuming the magic combination of transcription factors can be identified. "I think it's possible, and I would not be surprised in the next 12 months to see many examples published in the literature," he says.

That's not to say iPSCs are going anywhere, he adds. Neurons, as terminally differentiated cells, do not divide; thus, to create the quantities needed for clinical and drug development applications, the iPSC approach makes more sense, he says, as iPSCs proliferate freely. On the other hand, iPSC generation is "a laborious and lengthy process" requiring months of effort, and differentiation remains relatively inefficient, meaning the iN approach might be a better choice, for instance, when creating a battery of patient-specific neuronal lines, Wernig says.

Of course successful differentiation, by whatever means, is still just another step forward in a marathon race. Next up: demonstrating that these differentiated cells "can really model disease," says Zhang. "If we can achieve that, that's a great step."

References
1. K. Takahashi et al., "Induction of pluripotent stem cells from adult human fibroblasts by defined factors," Cell, 131[5]:861-72, 2007.

2. "Geron and FDA reach agreement on clinical hold," Geron Corp. press release, Oct. 30, 2009. [http://www.geron.com/media/pressview.aspx?id=1195]

3. L.G. Villa-Diaz et al., "Synthetic polymer coatings for long-term growth of human embryonic stem cells," Nat Biotechnol, published online May 30, 2010, doi:10.1038/nbt.1631.

4. Z. Melkoumian et al., "Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells," Nat Biotechnol, published online May 30, 2010, doi:10.1038/nbt.1629.

5. S. Rodin et al., "Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511," Nat Biotechnol, published online May 30, 2010, doi:10.1038/nbt.1620.

6. X. Carvajal-Vergara et al., "Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome," Nature, 465:808-12, 2010. doi:10.1038/nature09005.

7. B.-Y. Hu et al., " Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency," PNAS, 107:4335-40, 2010. Doi: 10.1073/pnas.0910012107.

8. T. Vierbuchen et al., "Direct conversion of fibroblasts to functional neurons by defined factors," Nature, 463:1035-41, 2010. doi:10.1038/nature0879.

9. Q. Feng et al., "Hemangioblastic derivatives from human induced pluripotent stem cells exhibit limited expansion and early senescence," Stem Cells, 28:704-12, 2010.

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