Techniques for generating human dopaminergic neurons: reprogramming and differentiation

Perhaps more than any other medical field, stem cell research could have a significant and wide-reaching near-term public health impact. Cell-replacement therapies and other treatments are in currently in development for over 50 diseases.

Recent advances have been achieved in techniques for generating human neurons that could help – both through reprogramming somatic cells directly to neurons, and in differentiating pluripotent cells to neurons.

• Reprogramming is converting cells from one type to another by introducing additional genes (e.g.; transcription factors) to transform them. Recent research makes use of both canonical and novel transcription factors, and the application of growth factors, microRNAs, and small molecules. This technique is particularly vaunted as it may be possible to reprogram human adult cells such as skin cells, which are easily attainable, into any other type of needed cell, such as heart cells and neurons.
• Differentiation is the other main technique for generating neurons. In this case, human pluripotent stem cells are differentiated to neural cells with the introduction of growth factors and detailed cell-culturing processes.

Neurons and neural stem cells are of particular translational interest due to the lack of effective clinical therapies for neurodegenerative disease. While these emerging techniques are promising, some of the processes are relatively new, and the fidelity and functionality of generated neurons and neural stem cells in clinical application is yet to be confirmed.

Steady advance of stem cell therapies

Stem cell research and related therapies (including regenerative medicine and cellular therapies) is an industry with a strong possibility of having a significant near-term impact on worldwide public health. One reason is the industry’s linkage between policy, science, industry, and patient advocacy, as was clear in the attendance and programming at the 7th annual World Stem Cell Summit held in Pasadena CA, October 3-5. Other science-driven fields such as synthetic biology, nanomedicine, and aging might benefit from cultivating such a multi-disciplinary perspective. Stem cell therapies are useful not only in cell-replacement therapies, but also in disease modeling, drug discovery, and drug toxicity screening.

Disease therapeutics and clinical trial focus
Stem cell therapies are currently being applied to over 50 diseases particularly in the areas of heart, lung, neurodegenerative, and eye disease, and cancer and HIV. Dozens of companies are developing therapeutic solutions which are in different stages of clinical use and clinical trials. Some high-profile therapies include Dendreon’s Provenge for prostate cancer, Geron’s first-ever embryonic stem cell trials for spinal cord injury, Fibrocell’s laViv cellular therapy for wrinkles, and well-established commercial skin substitutes (Organogenesis’s Apligraf and Advanced BioHealing’s Dermagraft).

Policy
Stem cell policy issues under consideration include medical tourism, standards for large-scale stem cell manufacturing, and lingering ethical debates over the use of embryonic stem cells.

Science
Contemporary stem cell science advances include a focus on techniques for the direct reprogramming of cells from one lineage to another without having to return to pluripotency as an intermediary step, improved means of generating and measuring induced pluripotent cells, and progress in approaches to neurodegenerative disease, for example establishing causal factors for early-onset Parkinson’s disease, generating neuronal cells and dopaminergic cells, and neural stem cell lumbar implantation clinical trials.

Regenerative medicine: conduits, augments, and blood-vessel printing

A medical advance that could have as large an impact on disease eradication and life extension as penicillin is regenerative medicine and tissue engineering. The field was perceived as revolutionary even a few years ago, but rapid advances have made it seem almost commonplace today.

Replacement organs grown from one’s own cells are desirable since this would avoid immune system rejection and a lifetime of immunosuppressive drugs and their side effects.

Hollow organs like the bladder are easier to create than the more solid liver and kidney, and the heart is the most challenging. The current status is that several dozens of lab-generated bladders have been implanted in humans.

In the case of other organs, the present focus of tissue engineering is on conduits (e.g.; providing a link to the outside of the body for waste removal (Tengion neo-urinary conduit clinical trials)) and augments (e.g.; providing a supplemental path for normal operations (Tengion neo-kidney augment and other augments)).

Vascularization, or blood-vessel printing, is an intense area of research focus, and there is some promising progress from at least two sources, well-known regenerative medicine research leader Anthony Atala, and tissue engineering startup darling Organovo. This could be a key step in allowing more sophisticated organs to be regenerated.

Status of Stem Cell Research

The World Stem Cell Summit in Baltimore MD held September 21-23, 2009 attracted several hundred professionals to discuss contemporary science, industry and societal perspectives on stem cells. Attendance was high, but down from last year and, similar to cancer meetings, a key theme several keynote speakers acknowledged was

the overall lack of truly meaningful progress in stem cell research in the last twenty years.

Science Focus: Safe Stem Cell Generation
The science tracks featured current research in different stem cell areas including the production of safe hESC (human embryonic stem cells) and iPS (induced pluripotent stem cells) for use in regenerative medicine, the research and therapeutic use of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) and reports from specific sub-fields: cancer stem cells, cardiovascular stem cells and neural stem cells. Overall, the work presented was incremental and in many cases, confirming what has been known already, such as a growing confirmation that cancer stem cells are probably responsible for triggering the resurgence of cancer but cannot at present be distinguished from other cells at the time of tumor removal.

Contract Research Demand: Cell Therapies and Recombinant Proteins
One stem cell area experiencing growth is contract research organizations, the outsourcing tool of choice for research labs and pharmaceutical companies in the production of biological materials. For large contract research manufacturing such as Basel, Switzerland-based Lonza, the biggest demand area is in cell therapies. Cell therapies denote the introduction of any type of new cell into other tissue for therapeutic purposes, but in the current case generally means any variety of stem cell-based therapies. Other large contract research manufacturing organizations such as Morrisville, NC-based Diosynth (owned by Schering Plough) lead in biologics (antibodies, protein production) production, an important area for nextgen biotech where synthetic biology could have a big impact.

For smaller contract research manufacturing organizations producing test compounds (e.g.; 1 liter for $10,000) and scaling to Phase I and II clinical trial quantities such as Baltimore MD-based Paragon Bioservices, the biggest demand is for recombinant proteins. Recombinant proteins are created by inserting recombinant DNA into a plasmid of rapidly reproducing bacteria and can take many useful forms such as antibodies, antigens, hormones and enzymes.

Venture capital hot topics: zinc fingers, RT PCR, tech transfer
Zinc fingers (small protein domains that bind DNA, RNA, proteins and small molecules) have been surfacing in a variety of cutting-edge biotech innovations. In July 2009, St. Louis, MO-based biotechnology chemical producer Sigma-Aldrich (SIAL) announced the creation of the first genetically modified mammals using zinc finger nuclease (ZFN) technology to execute modifications such as taking away the tail of the zebrafish. A second example of recent landmark research involving zinc fingers is that of Carlos Barbas at Scripps who uses zinc finger proteins to reprogram serine recombinases as a more specific alternative to the homologous recombination method of genome modification. In addition, the Barbas lab has a useful web-based zinc finger protein design tool available for public use, Zinc Finger Tools.

Real-time PCR offerings continue to expand and flourish with declining prices as startup newcomer Helixis announced a $10,000 real-time PCR solution at the conference.

Bethesda, MD-based Toucan Capital, a leading investor in stem cells and regenerative medicine discussed their sixteen interesting portfolio companies such as San Diego CA-based VetStem who is conducting joint and tendon stem cell therapies for race horses.

Johns Hopkins has one of the country’s leading technology transfer programs, licensing a growing number of technologies each year (nearly 100 in the last fiscal year), and has a searchable, though not extremely user-friendly, website.

Ethics of brainless humans

As a thought experiment, if it were possible, would it be ethical to make humans without brains for research purposes?

The idea arises since a more accurate model of humans for drug testing would be quite helpful. Drugs may work in mice, rats and monkeys but not in humans or in some humans but not others. Human biology is more complex and the detailed pathways and mechanisms are not yet understood.

Of course by definition, a brainless human is not really a human; a human form without a brain would be more equivalent to a test culture of liver cells than a cognitive agent.

Tissue culturing, regenerative medicine and 3D organ printing
The less contentious versions of the idea of growing brainless humans is currently under initial exploration in taking tissue from a human, growing it up in culture and testing drugs or other therapies on it. A further step up is regenerative medicine, producing artificial organs from a person’s cells such as the Wake Forest bladder and Gabor Forgacs 3D organ printing work.

Brain as executive agent may be required
The next steps for testing would be creating systems of interoperating tissue and organs (e.g.; how would this person’s heart and liver respond to this heart drug?) and possibly a complete collection of human biological systems sans brain. One obvious issue is that this might not even work since the brain is obviously a critical component of a human and that a brainless human could not be built, that some sort of executive organizing system like the brain would be needed. Also medical testing would need to include the impact on the brain and the brain’s role and interaction with the other biological systems and the drug.

Ethical but impractical
Where it is quite clear that generating a full living human for research purposes would be unethical, it is hard to argue that generating a brainless human, a complex collection of human biological systems without a brain, which is not really human and does not have consciousness or personhood, would be unethical. Certainly some arguments could be made to the contrary regarding the lack of specific knowledge about consciousness and concepts of personhood, but would seem to be outweighed.

Unlikely to arise
It is extremely unlikely that the situation of manufacturing brainless humans for research purposes would ever arise, first since a lot of testing and therapy may be possible with personalized tissue cultures and regenerative medicine, and informed by genomic and proteomic sequencing. Also, in an eventual era where it might be possible to construct a brainless human or a collection of live interacting tissues and organ systems, it would probably be more expedient to model the whole biological system digitally.

Aging is solvable

That aging is understandable and solvable, not necessarily immediately but ultimately, was one topic not seeing a lot of opposition at the American Aging Association (AGE) conference in Phoenix AZ May 29 – June 1, 2009. Key research highlights are below.

Aging is a key contemporary concern, on the order of climate change, as all countries worldwide have populations increasingly stratified towards aging. Aging is not just a medical condition but a key challenge to be resolved for advanced societies to be successful in the long-term. Productivity, healthcare costs and happiness and comfort could all be improved with advances in the remedy of aging. Aging has advanced from a nebulous concept to concrete mechanisms that can be understood and managed. Thematically, most of the bioparts impacted in aging (cells, genes, proteins, neurons, etc.) seem to still be present in older organisms, just not functioning the way they did when the organisms were younger, suggesting that it may be possible to manage and reverse aging processes, and confirming the systemic nature of aging including, for example, the role of a healthy microenvironment and cell-cell signaling. Reductionism as an approach has proved unsuccessful.

Aging is a multidisciplinary phenomenon, involving different deterioration processes in different tissues over time. Aging involves a variety of fields (immunology, cancer, regenerative medicine, cognition, micronutrients, etc.) and a variety of levels of research species (C. elegans (worms), Drosophila (flies), mice, rats and humans). At AGE, the organizational structure was a focus on systems pathways, particularly signaling and hormones, together with a look at the role of proteins in aging.

AGE was an excellent place to obtain a broad and deep comprehension of how aging works. The systemic rigor required to characterize the process-intensive nature of aging has been making significant progress, with a much more detailed understanding of the complex nested multifactor pathways now existing as compared with that of even a few years ago. It is clear that the painstaking characterization work could be further improved with automation and quantitative tools, especially for example, digital linkage of aging pathways across organisms.

As with other life sciences areas, the potential widespread quick and cheap availability of the sequencing of genomes, proteomes, etc. is likely to dramatically change how the science of aging is conducted, though not guarantee quick solutions. As pathways continue to be confirmed, they can be digitized into software and nearly indefinite simulated iterations could be run before conducting time-consuming and expensive bench experiments in confirmation.

Many interventions work for extending the lifespans and healthspans of lower order organisms, for example knocking out any one of 200 known genes may extend the lifespan of the C. elegans worm but the specifics and replicability of the mechanisms in higher order organisms are not known. It does not make sense to directly translate point solutions up to mammals given the systemic nature of the organisms and aging processes. Even moving one biomarker for alcohol consumption from monkeys to humans is not direct.

Exciting new research findings
Reference links below and conference abstracts here

1. 3-D organ printing: Use only biologics (cells and cell products) in a scaffold-free tissue engineering process to print 3-D tissues and organs which can be vascularized prior to implantation, relying on developmental biology to trigger the cells to fuse and self-assemble into organs. (Gabor Forgacs, video, lab, organ printing)
   
2. Stem cell antibodies: Improve existing cardiac stem cell therapies (only 1% of cells reach the intended destination) by using specific antibodies for better targeting and retention of stem cells at sites of tissue injury. Replace cardiomyocytes with adult stem cells. (Jim Larrick, paper, general information)
   
3. Stem cells: Amplify and rejuvenate adult stem cells for injection into knees and hips as an alternative to surgical replacements. (Regenexx)
   
4. Bioremediation: Use natural enzymes to remediate biological build-ups; cholesterol oxidase from Brevibacteria to reduce 7KC cholesterol in atherosclerosis and A2E-degrading enzymes to improve macular degeneration. (John Schloendorn, research program, paper)
   
5. Life extension: Examine the mechanisms of dietary restriction (DR) with further elucidation of TOR (target of rapamycin) pathways, a fast growing area of research. Find that inhibiting a downstream gene in the TOR pathway, HIF-1 (a transcription factor important for growth and metabolism), extends lifespan in worms. (Pankaj Kapahi, paper)
   
6. Life extension: Generate a 10x lifespan extension in C. elegans by silencing many components of insulin/IGF-1 signaling (IIS) possibly via the disruption of PIP3 (a key signaling molecule required for the membrane tethering of many signaling molecules). (Puneet Bharill, paper)
   
7. Amyloid plaque reduction: Use a known plaque imaging agent, ThT (Thioflavin T), as a therapeutic for amyloid plaques. (Silvestre Alavez, lab affiliation, paper)
   
8. Cancer protection: Find that naked mole rats have two layers of anti-cancer protection, humans have only one. p16 is the first-line-of-defense anti-cancer protection mechanism found in naked mole rats. Humans (and other organisms) also have p16 (a suite of three genes), perhaps the mechanism for its upregulation (probably a cell:cell signaling dynamic) in naked mole rats could be understood and turned on with an enzyme in humans. (Andrei Seluanov, earlier research)
   
9. Cognitive function: Find that neurogenesis is possible in aged organisms with exercise followed by cognitive stimulation (e.g.; tackling a puzzle or challenge); organisms can benefit by building up a larger reservoir of brain cells earlier in life by being exposed to a variety of external stimulation. (Gerd Kempermann) This author’s speculation: Perhaps neurogenesis could be further harnessed for brain enhancement beyond currently realizable human capacities as this mechanism is better understood.
   
10. Aging biomarkers: Upstream the aging focus to prevention by measuring biomarkers and introducing interventions. Some suggested biomarkers of aging are p16 gene levels (which can be decreased with exercise), telomere length, the level of senescent cells, and the number of circulating lymphocytes in the immune system (measure total T cells (CD3+), B cells (CD19+) and CD28 absolute numbers on CD8+ T cells). (Kronos research projects, test menu; telomere length measuring)
    
11. Hormones-IGF: Find no conclusive evidence of insulin-like growth factor's (IGF) ability to retard natural aging, though on an individual basis some people may find it useful. (Marc Blackman)
    
12. Hormones-HRT: Find that hormone replacement therapy (HRT) can be good for improving cognitive function and bone loss in women that do not have a risk of heart disease; HRT should be started with the onset of menopause, not later. (Barbara Sherwin, Eef Hogervorst)
    
13. Cost of reproduction: Find that ovary removal in grasshoppers resulted in a 25% increased lifespan, contributing to existing evidence regarding the high cost of reproduction. (John Hatle) This author’s speculation: In the farther future, in humans, it could be quite desirable to closely manage fertility, turning it on and off at will, if fertility is even necessary.
    
14. Micronutrients: Find tremendous nutritional benefits from the consumption of fruits with skin, especially blueberries (pterostilbene that reduces oxidative stress), blackberries, raspberries, red grapes, pomegranates, cranberries, plums, strawberries, cherries, pears and apples (phytochemicals that provide cancer prevention), walnuts (preventing the inflammation and oxidative stress of brain aging:), green tea (catechins that reduce cardivascular and cancer risk) and tempeh (fermented whole soy bean with folate is healthier than tofu (processed soy bean curd)). (Blueberries: Agnes Rimando, Rolf Martin; Apples: Rui Hai Liu, Walnuts: James A. Joseph, Green tea: Vojo Deretic, Tempeh: Eef Hogervorst)
    
15. Calorie restriction (CR)/dietary restriction (DR): Find that in humans, improved biomarkers for CR/DR, vegan and raw food diets that result in the extension of the onset of aging challenges. (John Holloszy)
    
16. Aging mice testbed: A mouse type that sufficiently recapitulates early aging, the human WS phenotype (Werner syndrome), has been created which could hasten mammalian aging research. (David Kipling)
    

Conclusion
Aging is a key contemporary issue. Research is advancing both incrementally and radically in every area of aging. The highest immediate impact could come from working on aging problems upstream at important fulcrum points that impact everything below them, such as genetics, epigenetics and the immune function. The research is progressing and it is starting to be time for VCs, big pharma and DIYbio’ers to take advantage of the many interesting and actionable possibilities.