Genes Cut Out of Reprogrammed Cells

Lots of people (here, here, and here, etc.) are commenting on the "Proof of Concept" by Jaenisch, et. al., in this week's ScienceExpress (early online publication before print) that showed gene modification to reprogram mouse cells in order to create blood line stem cells that would achieve gene therapy - or even, a cure - for sickle cell anemia.

(BTW, these mice are called "humanized knock in mice," meaning that the genes of the have been modified so that the their bone marrow hematopoietic or blood line stem cells have genes "knocked in" to produce human cells blood cells.)

To reduce the potential risk of tumor formation due to c-Myc transgene expression (13), iPS cells were infected with an adenovirus encoding Cre-recombinase to delete the lentivirus transduced c-Myc copies. One out of 10 iPS subclones (iPS #3.3) had deleted both transduced copies of c-Myc and was used for further experimentation (Fig. 2C).

Trust me, as soon as more labs figure out how to make use of these cells, to remove or repair - or to ensure there's no - damage from the insertion of the needed genes, the push for embryos will slow down. (I think it would even faster if Thomson and the California Institute for Regenerative Medicine could find a way to capitalize on adult stem cell research.)

Flash: Embryonic tissue more difficult to obtain than adult

This could liberate future researchers from relying on embryonic tissue, which can be more difficult to acquire.

"NatureNews," the news alert website for the journal Nature, has a news report (registration required) on a study published in the December 6 issue of the journal by researchers from Bonn, New York, Rhode Island, and Pennsylvania on regenerating heart muscle.

It is known that (in mice and assumed in humans) that embryonic heart stem cells or precursor cells (they're a bit more specialized than most of the "stem cells" we've been hearing about - cardiac myocytes (eCMs) and skeletal myocytes ("SMs") will bind to damaged areas of the heart and promote healing and division. However, the SMs have a much higher risk of irregular heartbeats called "ventricular tachycardia." (The "VTach" that causes most sudden deaths after heart attacks in real life and the dramatic scenes on TV.) Even though some people do have better heart function after treatment with their own SMs (from bone marrow or skeletal muscle), 15% of them die of VTach within 3 years.

The researchers found that the eCM's "coupled physically" to the damaged cells in the heart and exchanged electrical signals with the surrounding heart cells, so that they contracted in the proper rhythm.

The scientists (rather than deciding to pursue only eCM therapy) wanted to know why the SMs didn't do as well as eCM when it came to producing the correct rhythm. They discovered that the eCMs had more Cx43 than skeletal muscle. Mice were developed with gene therapy (using viral vectors to insert genes, a common technique used to create "gene mod" mice for research) so that the SMs of the mice express more Cx43 in skeletal muscle than normal. The mice hearts that received the modified SM's from these mice, when cultured and then injected into damaged hearts of other mice, did as well as the hearts that received eCMs.

The science is fascinating, but the irony of the report coming this week is pretty interesting, too. The author of the NatureNews report is truly unbiased or evidently didn't get the memo from the reactionary scientists this week.

(In case you're wondering, I don't get a memo from anyone other than the reactionaries' own blogs, and statements to the press.)

Wash this reactionary's mouth out with soap!

Bioethics.net compares the Bush administration's happiness about reprogrammed adult stem cells with that man, Mr. Clinton's, "I did not have sex with that woman!" and President Bush's statement "Mission accomplished," after our US troops captured Baghdad.

I'll accept the latter (at some future date, if the evidence supports it), but the first is at least as false as Clinton's wagging finger - and (speaking of Yuk factors) did we really need to be reminded of that?

The author, James W. Fossett (who is anything but "non-partisan") states that Yamanaka, the first to report reprogrammed adult cells in humans and mice is from Japan and wasn't affected by the US Federal funding limitations. He doesn't mention that Yamanaka's research didn't rely on the use of new embryonic cells, at all. Yamanaka took the information gleaned from animal research and the currently funded cells and moved to the front of all other stem cell researchers by pointing the way to the key to the production of stem cells from each patient who needs them - from his or her own cells.

Instead, Fossett is running scared due to the "rhetorical parity" from cell reprogramming and the possibility that the success in reprogramming cells will result in more reprogramming research!

Fossett doesn't mention that James Thomson's research using human Embryonic Stem cells (hESC). then human fetal cells harvested after abortions - and finally in skin cells harvested at circumcision of little boys - was funded by the National Institutes of Health, and that those hESC are the ones that supposedly are of no use.

Fossett also fails to mention of the new report by Yamanaka on the technique using only 3 inserted genes to the prior 4, and that the eliminated gene is the one that had scientists concerned about cancers.

I'm sure that he doesn't recall the "first transplantable lung cells" from hESC's by Texas researchers last year. These cells were developed by viral "transfection," also, and were lauded as "a platform that could potentially be useful in the development of spinal cord cells, heart cells, nerve cells and others.” These were neither the first or transplantable, but they did get much more notice than similar cells developed from umbilical cord blood cells without viral transfection.

That may be the problem: the proponents of hESC research are used to getting many times the publicity from hESC research than that received by the non-hESC researchers. And so, we get the concerns about "rhetoric."

There's those deceitful knee jerk reactionaries practicing their projection, again.

Dolly's Dad: Cloning, embryos and eggs not needed

10 years after the world learned about the cloning of Dolly the sheep, the scientist responsible for her birth announces that cloning is passe'.

Just after the announcement that a US lab has managed the first confirmed cloning of primate (monkeys, not human) embryos using adult cell donor DNA, Ian Wilmut made statements to the UK press that he's abandoning the cloning track for research using something like Japan's Yamanaka's process of dedifferentiation or reprogrammed adult cells to produce stem cells. (He's also selling the paperback version of his latest book.)

This approach, he says, represents, the future for stem cell research, rather than the nuclear transfer method that his large team used more than a decade ago at the Roslin Institute, near Edinburgh, to create Dolly.

Last year, at the American Society of Bioethics and Humanities Conference, the top ‘ethicists’ were nearly in a panic over these techniques. They evidently put pressure on Yamanaka to and criticize his own research. Then, 3 other labs proofed the technique and the Yamanaka’s lab advanced a step or two. Gearhart and Moreno were still scoffing last month, when they spoke at the National Academies of Sciences museum during the ASBH conference.


I’m afraid that too many labs and too many PhD candidates and sponsors have all their eggs in the cloning basket for the issue to fade decently. Talk about being left behind — all US research centers, such as the California Institute of Regenerative Medicine — will be negated if they insist on following the dead end trail of cloning and unethical destructive embryonic stem cell research.

The Yamanaka technique involves reprogramming adult fibroblasts - skin precursors - to a primitive, embryonic stem cell state. The stem cells are not quite 'totipotent" from what we can tell -- they aren't capable of forming new embryos. But they are capable of forming "all the cells of the body," at least with manipulation in the proper environment.

We're going to hear more and more dispute about the "proper" name for the cells -- disputes over whether they are actually stem cells. And a huge amount of discussion about the dangers from the viral transfection that is used to add the genes that turn on stemness.


We will be expected to forget that

1. No one has been able to clone a human embryo in spite of thousands of eggs used in Hwang's scam alone,

2. That 13,000 monkey eggs were used in the latest attempt to clone primates, that the published study relates an efficiency of 0.7% success, and the authors aren't quite sure why they were successful where other labs weren't,

3. The fact that true embryonic stem cells are short lived in the body and difficult to control (reports actually criticized the embryonic like stem cells from dedifferentiation for making tumors in mice - although that is one of the properties that defines embryonic stem cells)

4. That transfection with plasmids and specialized virus particles is an established technique of gene therapy,

5. and that the production of stem cell lines toward the end-stage adult cells has used viral transfection as well.

Even though he spouts the proper mumbo jumbo about the "moral status of the human embryo" (and that a person is someone capable of valuing herself - with the gradual acquisition and loss connected with functional capability), don't be surprised if Wilmut is, himself, negated and Watsoned because of his disloyalty to the cloning and embryo-destructive catechism. (James Watson's non-PC comments from last month cost him his lab, although he's been saying the same things for years.)


Now, if only the US, and especially the Texas, research Powers That Be will pay attention and learn.

Umbilical Cord vs. Embryonic Stem Cells

The Proceedings of the National Academies of Science (PNAS) has published the article that was the subject of this blog last week, and which claims that researchers at the University of Texas at Houston have produced the "first transplantable source of lung epithelial cells." There is no evidence that these cells are "transplantable," and they are definitely not the first team to produce Alveolar Type II ("ATII") lung cells from more primitive stem cells. The article does an excellent, if technical, job of explaining the importance of ATII lung epithelial cells:

The alveolar epithelium covers [approximately] 99%of the internal surface area of the lung and is composed of two major cell types, the alveolar type I (ATI) cell and the alveolar type II (ATII) cell. ATI cells are large flat cells through which exchange of CO2/O2 takes place. They cover [approximately] 95% 0f the alveolar surface and comprise [approximately] 40% of the alveolar epithelium and 8% of the peripheral lung cells. In contrast, ATII cells are small, cuboidal cells that cover [approximately] 5% of the alveolar surface and comprise 60% of the alveolar epithelium and 15% of the peripheral lung cells. They are characterized by the unique ability to synthesize and secrete surfactant protein C (SPC) and by the distinct morphological appearance of inclusion bodies, known as lamellar bodies. Important functions of ATII cells are (i) to synthesize, store, and secrete surfactant, which reduces surface tension, preventing collapse of the alveolus; (ii) to transport ions from the alveolar fluid into the interstitium, thereby minimizing alveolar fluid and maximizing gas exchange; (iii) to serve as progenitor cells for ATI cells, which is particularly important during reepithelialization of the alveolus after lung injury; and (iv) to provide pulmonary host defense by synthesizing and secreting several complement proteins including C3 and C5 (1–3) as well as numerous cytokines and interleukins that modulate lymphocyte, macrophage, and neutrophil functions (4). Severe pulmonary diseases can be caused by deficiencies or genetic mutations in proteins synthesized by ATII cells that are important in maintaining normal lung homeostasis. For example, complete deficiency of surfactant protein B (SPB) is caused by genetic mutations in the SPB gene. This deficiency results in impaired pulmonary surfactant composition and function and is a major cause of fatal neonatal respiratory disease (5, 6). In addition, ATII cells synthesize and secrete the serine protease inhibitor alpha-1-antitrypsin (alpha-1 AT) which also plays a key role in alveolar homeostasis by regulating protease imbalance and adjusting fluid clearance (7, 8), the importance of which is supported by the association of alpha-1 AT deficiency with the development of pulmonary emphysema (9). Cystic fibrosis is thought to be primarily a disease of the upper airway and submucosal epithelia and is caused by mutations in the cystic fibrosis transmembrane conductance receptor (CFTR) (10). CFTR is an important regulator of Cl and liquid transport in the lung (11–14) and is functionally expressed by human ATII cells, strongly suggesting a critical role for CFTR in regulating ion and fluid transport in the lung alveolus in addition to the upper airway (13).

(The numbers in parentheses refer to footnotes. Also, I had to change some of the characters to words: "alpha" and "approximately")

The PNAS report and UT Houston's Press Release do not contain any note about the earlier umbilical cord blood stem cell research, although the latter was published on line and in print in Cytology, at least 2 weeks before the initial submission of the PNAS article.

Both research teams report that they followed the techniques developed and reported in the lab of another researcher, Samadikuchaksaraei, in growing, multiplying and guiding the differentiation of their primitive cells toward the more specialized lung cells that were desired. Both report the successful production of ATII lung cells, as demonstrated by the way the cells look and by demonstrating the production of Surfactant Protein C - which, in human development is only found in mature ATII cells after the unborn (or premature) child has reached 36 weeks of gestation.

The Houston team claims that one reason their process is superior to the earlier Embryonic Stem Cell research is that they were able to produce mature cells in 10 days, while Samadikuchaksaraei's team took 15 days. If the ability to produce the cells in what Wetsel, et. al., describe as a "timely manner," then it is important to note that the Minnesota team produced their mature ATII cells in 3 to 8 days.

The Houston team also claims that is possible that their new cell lines might one day be transplanted, although there has never been any research reporting the successful transplantation of epithelial cells into the lungs. Another problem is that the cells were guided to change by "transfection" with a segment of DNA that is inserted into the genes of the cells, using a retrovirus. Any use of these cells, even if anyone ever proves that we can transplant cells into the lung and cure a disease, will be complicated by years of research to prove that the gene therapy that produced these cells is safe and stable in the lungs of the patients. The authors do not give us any references to support this hypothesis.

From the Discussion section of the PNAS article:

"Lung injury due to chronic pulmonary diseases, such as chronic
obstructive pulmonary disease and asthma, and inherited genetic disorders, such as cystic fibrosis and
1-AT deficiency are leading causes of morbidity and mortality worldwide. Cystic
fibrosis and
1-AT deficiency are two of the most common inherited genetic defects affecting Caucasians. In addition, SPB deficiency is a major cause of respiratory disease and fatality in neonates. All three of these diseases are caused by single-gene defects and therefore have been logical candidates for gene therapy. However, efficient vector delivery and sufficient transgene expression needed for therapeutic benefit have remained elusive. Recent research advances indicate that gene delivery via transplantation of cells derived from human stem cells may provide an attractive alternative to viral or liposome vector based gene therapies. Moreover, transplantation of cells derived from human stem cells may prove ideal for the repair and regeneration of injured lung tissue.

Because of its ability to proliferate as well as to differentiate into ATI cells, the ATII cell is an excellent choice of lung cell for possible therapeutic use in gene delivery and repair of the alveolus.

. . . The use of ES cells as a source of transplantable cells in the lung alveolus will require the generation of significant quantities of highly pure ATII cells. To achieve this goal, we chose to genetically modify hES cells so that resulting differentiated ATII cells could be enriched through antibiotic selection. Our approach was to establish a stable transfected hES cell line containing a single copy of the human SPC promoter-Neor fusion gene. When subjected to differentiation in vitro, it was hypothesized that ATII cells derived from this genetically modified hES cell line (SPCP/NEO.74) would express the Neor gene and would therefore survive G418 antibiotic selection, whereas, all of the other differentiated cell lineages as well as the pluripotent cells would be eliminated by G418 selection. Immunocytochemical and flow cytometric analysis of the surviving G418-selected cells supported this hypothesis, indicating that this genetic selection approach resulted in an enrichment of hES-ATII cells to 99% when cultured on Matrigel-coated plates. Our protocol reproducibly produced from each 10-cm culture dish 106 essentially pure ATII cells within 15 days of differentiation. These differentiated ATII cells survive for at least 2 days in culture in the absence of G418 and will provide in a timely manner sufficient numbers of pure ATII cells for future transplantation investigations."

(No footnote references were removed from this quoted portion.)


The abstracts are available on line for free, but the actual articles are available only by subscription or by paying for temporary access:

From Proceedings of the National Academies of Science, published online March 2, 2007

"A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells"

Dachun Wang, David L. Haviland, Alan R. Burns, Eva Zsigmond, and Rick A. Wetsel. Research Center for Immunology and Autoimmune Diseases and Laboratory for Developmental Biology, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center; Department of Biochemistry and Molecular Biology, University of Texas Medical School; and Cardiovascular Sciences Section, Department of Medicine, Baylor College of Medicine. Communicated by C. Thomas Caskey, University of Texas Health Science Center, Houston, TX, January 4, 2007 (received for review November 22, 2006)

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.
Keywords: complement , differentiation, surfactant proteins, alpha-1-antitrypsin, cystic fibrosis transmembrane conductance receptor

And here's the abstract of the report from November in Cytotherapy, (2006) Vol. 8, No. 5, 480-48 (Note the association with BioE, Inc., the company that's doing the cancer research with MD Anderson):

"Differentiation of umbilical cord blood-derived multilineage progenitor cells into respiratory epithelial cells."

MJ Berger, SD Adams, BM Tigges, SL Sprague, X-J Wang, DP Collins and DH McKenna, Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota, USA, Clinical Cell Therapy Laboratory, University of Minnesota Medical Center, Minneapolis, Minnesota, USA, and BioE Inc., Saint Paul, Minnesota, USA

Background - Umbilical cord blood (UCB) has been examined for the presence of stem cells capable of differentiating into cell types of all three embryonic layers (i.e. endo-, ecto- and mesoderm). The few groups reporting success have typically confirmed endodermal potential using hepatic differentiation. We report differentiation of human UCB-derived multipotent stem cells, termed multilineage progenitor cells (MLPC), into respiratory epithelial cells (i.e. type II alveolar cells).
Methods - Using a cell separation medium (PrepaCyte-MLPC; BioE Inc.) and plastic adherence, MLPC were isolated from four of 16 UCB units (American Red Cross) and expanded. Cultures were grown to 80% confluence in mesenchymal stromal cell growth medium (MSCGM; Cambrex BioScience) prior to addition of small airway growth medium (SAGM; Cambrex BioScience), an airway maintenance medium. Following a 3 - 8 day culture, cells were characterized by light microscopy, transmission electron microscopy, immunofluorescence and reverse transcriptase (RT)-PCR.
Results - MLPC were successfully differentiated into type II alveolar cells (four of four mixed lines; two of two clonal lines). Differentiated cells were characterized by epithelioid morphology with lamellar bodies. Both immunofluorescence and RT-PCR confirmed the presence of surfactant protein C, a protein highly specific for type II cells.
Discussion - MLPC were isolated, expanded and then differentiated into respiratory epithelial cells using an off-the-shelf medium designed for maintenance of fully differentiated respiratory epithelial cells. To the best of our knowledge, this is the first time human non-embryonic multipotent stem cells have been differentiated into type II alveolar cells. Further studies to evaluate the possibilities for both research and therapeutic applications are necessary.
Keywords - endodermal differentiation, respiratory epithelium, stem cells, umbilical cord blood.