Forming Induced Pluripotent Stem Cells Inside a Living Organism


A team from the Spanish National Cancer Research Centre (CNIO) has become the first research team to convert adult cells that are still within a living organism into cells that show characteristics of embryonic stem cells.

The CNIO researchers also say that these embryonic stem cells, which were obtained directly from inside an organism, have a broader capacity for differentiation than those obtained by means of an in vitro culture system. Specifically, they have the characteristics of totipotent cells, a primitive state never before obtained in a laboratory, according to the CNIO team.

Manuel Serrano, Ph.D., director of CNIO’s Molecular Oncology Program and head of the Tumor Suppression Laboratory, led this study. It was supported by Manuel Manzanares, Ph.D., and his team from the Spanish National Cardiovascular Research Centre.

The CNIO researchers say their work extends that of Nobel Prize winner Shinya Yamanaka, M.D., Ph.D., one step forward. Yamanaka opened a new horizon in regenerative medicine when, in 2006, he demonstrated that stem cells could be created from adult cells by using a cocktail of genes. But while Yamanaka induced his cells in culture in the lab (in vitro), the CNIO team created theirs directly in mice (in vivo). Generating these cells within an organism brings this technology even closer to regenerative medicine, they say.

In a study published online Sept. 11 in the journal Nature, the CNIO research team details how it used genetic manipulation techniques to create mice in which Dr. Yamanaka’s four genes could be activated at will. When these genes were activated, they observed that the adult cells were able to de-differentiate into embryonic stem cells in multiple tissues and organs.

María Abad, Ph.D., lead author of the article and a researcher in Dr. Serrano’s group, said, “This change of direction in development has never been observed in nature. We have demonstrated that we can also obtain embryonic stem cells in adult organisms and not only in the laboratory.”

Dr. Serrano added, “We can now start to think about methods for inducing regeneration locally and in a transitory manner for a particular damaged tissue.” Stem cells obtained in mice also show totipotent characteristics never generated in a laboratory. Totipotent cells can form all the cell types in a body, including the placental cells. Embryonic cells within the first couple of cell divisions after fertilization are the only cells that are totipotent.

The researchers reported that they were also able to induce the formation of pseudo-embryonic structures in the thoracic and abdominal cavities of the mice. These pseudo-embryos displayed the three layers typical of embryos (ectoderm, mesoderm, and endoderm), and extra-embryonic structures such as the vitelline membrane, which surrounds the egg, and even signs of blood cell formation, which first appears in the primary embryonic vesicle (otherwise known as the “yolk sac”).

“This data tell us that our stem cells are much more versatile than Dr. Yamanaka’s in vitro inducted pluripotent stem cells, whose potency generates the different layers of the embryo but never tissues that sustain the development of a new embryo, like the placenta,” the CNIO researcher said.  Below is a figure from their paper.  The pictures look pretty convincing.

a, Cysts in the abdominal cavity of a reprogrammable mouse. b, Frequency of embryo-like structures after intraperitoneal injection of in vivo iPS cells (3 clones), in vitro iPS cells (2 clones) and ES cells (JM8.F6). Fisher’s exact test: *P < 0.05. c, Cyst generated by intraperitoneal injection. Left panels, germ layer markers: SOX2 (ectoderm), T/BRACHYURY (mesoderm) and GATA4 (endoderm). Right panels, extraembryonic markers: CDX2 (trophectoderm), and AFP and CK8, both specific for visceral endoderm of the yolk sac. d, Cyst generated by intraperitoneal injection presenting TER-119+ nucleated erythrocytes and LYVE-1+ endothelial cells in structures resembling yolk sac blood islands.
a, Cysts in the abdominal cavity of a reprogrammable mouse. b, Frequency of embryo-like structures after intraperitoneal injection of in vivo iPS cells (3 clones), in vitro iPS cells (2 clones) and ES cells (JM8.F6). Fisher’s exact test: *P < 0.05. c, Cyst generated by intraperitoneal injection. Left panels, germ layer markers: SOX2 (ectoderm), T/BRACHYURY (mesoderm) and GATA4 (endoderm). Right panels, extraembryonic markers: CDX2 (trophectoderm), and AFP and CK8, both specific for visceral endoderm of the yolk sac. d, Cyst generated by intraperitoneal injection presenting TER-119+ nucleated erythrocytes and LYVE-1+ endothelial cells in structures resembling yolk sac blood islands.

The researchers emphasize that any possible therapeutic applications of their work are still distant, but they believe that it could mean a change of direction for stem cell research, regenerative medicine and tissue engineering.

“Our stem cells also survive outside of mice in a culture, so we can also manipulate them in a laboratory,” said Dr. Abad. “The next step is studying if these new stem cells are capable of efficiently generating different tissues such as that of the pancreas, liver or kidney.”

This paper is very interesting, but I find it rather unlikely that their approach will take regenerative medicine by storm.  Engineering mice to express these four genes in an inducible manner caused the formation of unusual tumors throughout the mice.  Maybe they can be coaxed to differentiate into kidney or heart muscle or whatever, but learning how to get them to do that will take a fair amount of in vitro work.  This is interesting, but I doubt that it will change the field overnight.

RNA Molecule Protects Stem Cells During Inflammation


During inflammation and infection, bone marrow stem cells that make blood cells (so-called hematopoietic stem cells or HSCs) and progenitor cells are stimulated to proliferate and differentiate into mature immune cells. This especially the case for cells of the so-called “myeloid lineage.

Hematopoietic Stem Cells (HSCs) are able to differentiate into cells of two primary lineages, lymphoid and myeloid. Cells of the myeloid lineage develop during the process of myelopoiesis and include Granulocytes, Monocytes, Megakaryocytes, and Dendritic Cells. Circulating Erythrocytes and Platelets also develop from myeloid progenitor cells.

Hematopoiesis from Multipotent Stem Cell

Repeated infections and inflammation can deplete these cell populations, which leads to serious blood conditions and increased incidence of cancer.

A research team from the California Institute of Technology, led by Nobel Prize winner, David Baltimore, has discovered a small RNA molecule called microRNA-146a (miR-146a) that acts as a safety valve to protect HSCs during chronic inflammation. These findings also suggest that deficiencies for miR-146a might contribute to blood cancers and bone marrow failure.

Baltimore and his colleagues bred mice that lacked miR146a. MicroRNAs are very short RNA molecules (around 22 base pairs long) that regulate the activities of other genes. They control the expression of genes at the transcriptional and post-transcriptional level. In the case of miR146a(-) mice, whenever these mice were subjected to chronic inflammation, the total number and quality of their HSCs declined steadily. In contrast, miR-146a(+) mice were better able to maintain their levels of HSCs despite long-term inflammation.

The lead author of this work, Jimmy Zhao, said, “This mouse with genetic deletion of miR146a is a wonderful model with which to understand chronic inflammation-driven tumor formation and hematopoietic stem cell biology during chronic inflammation.”

Zhao also noted the surprising result that the deletion of one microRNA could cause such a profound and dramatic pathology. This underscores the critical and indispensable function of miR-146a in protecting the quality and longevity of HSCs. This work also establishes the connection between chronic inflammation and bone marrow failure and diseases of the blood.

Even more exciting is the prospect of synthesizing anti-inflammatory drugs that could treat blood disorders. In fact, it is possible that artificially synthesized miR146a might be an effective treatment if small RNAs can be effectively delivered to specific cells.

Zhao also noted the close resemblance that this mouse model has to the blood disorder human myelodysplastic syndrome or MDS. MDS is a form of pre-leukemia that causes severe anemia and a dependence on blood transfusions. MDS usually leads to acute myeloid leukemia. Further study of Zhao and Baltimore’s miR146a(-) mouse might lead to a better understanding of MDS and potential new treatments for MDS.

David Baltimore, senior author of this paper, said, “This study speaks of the importance of keeping chronic inflammation in check and provides a good rationale for broad use of safer and more effective anti-inflammatory molecules. If we can understand what cell types and proteins are critically important in chronic-inflammation-driven tumor formation and stem cell exhaustion, we can potentially design better and safer drugs to intervene.”

See Jimmy L Zhao, Dinesh S Rao, Ryan M O’Connell, Yvette Garcia-Flores, David Baltimore. MicroRNA-146a acts as a guardian of the quality and longevity of hematopoietic stem cells in mice.  DOI: http://dx.doi.org/10.7554/eLife.00537Published May 21, 2013.  Cite as eLife 2013;2:e00537.

Postscript: This paper is especially meaningful to me because my mother died of MDS. The fact that a better model system for MDS has been established is an essential first step in finding a treatment for this killer disease.

John Gurdon Embraces Human Cloning


Wesley Smith has reported that Nobel Laureate John Gurdon, who shared the Nobel Prize in Medicine this year with Japanese induced pluripotent stem cell discoverer Shinya Yamanaka, has come out in favor of human cloning.

From the story in the Daily Mail:
‘I take the view that anything you can do to relieve suffering or improve human health will usually be widely accepted by the public – that is to say if cloning actually turned out to be solving some problems and was useful to people, I think it would be accepted,’ he said. During his public lectures – which include speeches at Oxford and Cambridge Universities – he often asks his audience if they would be in favour of allowing parents of deceased children, who are no longer fertile, to create another using the mother’s eggs and skin cells from the first child, assuming the technique was safe and effective.

‘The average vote on that is 60 per cent in favour,’ he said. ‘The reasons for “no” are usually that the new child would feel they were some sort of a replacement for something and not valid in their own right. ‘But if the mother and father, if relevant, want to follow that route, why should you or I stop them?’

 

Smith then quotes from his magnificent book “Consumers Guide to a Brave New World,” which all my readers to RUN out to buy and read over and over again:

Scientists would have to clone thousands of embryos and grow them to the blastocyst stage [one week] to ensure that part of the process leading up to transfer into a uterus could be “safe,” monitoring and analyzing each embryo, destroying each one in the process. Next, cloned embryos would have to be transferred into the uteruses of women volunteers [or implanted in an artificial womb]. The initial purpose would be analysis of development, not bringing the pregnancy to a live birth. Each of these clonal pregnancies would be terminated at various points of development, each fetus destroyed for scientific analysis. The surrogate mothers would also have to be closely monitored and tested, not only during the pregnancies but also for a substantial length of time after the abortions.

Finally, if these experiments demonstrated that it was probably safe to proceed, a few clonal pregnancies would be allowed to go to full term. Yet even then, the born cloned babies would have to be constantly monitored to determine whether any health problems develop. Each would have to be followed (and undergo a battery of tests both physical and psychological) for their entire lives, since there is no way to predict if problems [associated with gene expression] might arise later in childhood, adolescence, adulthood, or even into the senior years.

 

Smith, in my view, is spot on. Therapeutic cloning will not stop at using cloned blastocysts to make patient-specific embryonic stem cell lines. The reason for this is that even though cells made from differentiated embryonic stem cells can have therapeutic value, such cells can also be rejected by the immune system of the host animal. A much more fail-safe way to do this experiment is to gestate the embryos to the fetal stage and use the fetal tissues.

Once we go down the road of cloning and destroying embryos just to make embryonic stem cell lines from them, what’s to keep us from aborting fetuses just to get their cells? This slippery slope is real and speaks volumes, none of it good, about a society that sacrifices its youngest and more vulnerable members to serve the needs of others. It cheapens human life to the nth degree and at its lowest point, it simple murder.

Gurdon, however, speaks of reproductive cloning to replace children lost through tragedy. While I can appreciate the sentiment, sentiment is an extremely poor reason basis for ethics. Folks, biology is not destiny. Cloning experiments in animals have shown us that even cloned embryos made from material taken from the same mother, that are genetically identical are neither identical to their mothers nor are they identical to each other. Random events that occur during development and the way each individual responds to their environment shapes them in a unique manner. The cloned sheep Dolly was completely unlike her cloned siblings in personality, behavior, or overall appearance. The same can be said for CC (for “Carbon Copy”), the first cloned cat, which looked unlike her mother and had a very different personality.

Yet these cloned children are asked from the second they are born to replace another child who is unlike them. The cloned child is a human person and while the right for each person to be authentically who there are in an inherent right of all human beings, this very right is denied these cloned kids – they are born for the very reason that they can be someone else. This is a violation of everything it means to be human, and it is the very reason no good thing can come from human cloning.

Gurdon is a brilliant scientist, but as we have seen before, great scientists sometimes make terrible ethicists.

Nobel Prize Goes To Stem Cell Scientists


The Nobel Prize in physiology or medicine this year has been awarded to a British researcher named John Gurdon and a Japanese stem cell pioneer named Shinya Yamanaka. Both researchers showed that adult cells in our bodies have all the genes necessary to be reprogrammed into a more developmentally primitive state. Their discoveries, scientist hope, may be the basis for new treatments for diseases like Parkinson’s, diabetes and for studying the roots of diseases in the laboratory.

The Nobel prize committee at Stockholm’s Karolinska Institute said the discovery has “revolutionized our understanding of how cells and organisms develop.”

In 1962, John Gurdon showed that DNA from mature cells such an intestinal cells or skin cells could be used to generate new tadpoles. This revolutionary experiment definitely demonstrated that the genome of adult cells still possessed all the genes necessary to drive the formation of all cells of the body.

Ian Wilmut’s work at the Roslin Institute in Scotland that culminated in the cloning of Dolly the sheep utilized the same process that Gurdon had designed in frogs and showed that it could work in mammals.

Gurdon did his landmark experiment in 1962, the year the other Nobel prize winner, Shinya Yamanaka, was born. More than 40 years after Gurdon’s discovery (2006), Yamanaka used an incredibly simple yet elegant recipe to convert mature cells into primitive cells that could be differentiated into different kinds of mature cells.

These primitive cells, induced pluripotent stem cells or iPSCs were equivalent to embryonic stem cells. Embryonic stem cells, however, were embroiled in controversy, since derivation of embryonic stem cell lines required the destruction of human embryos. However, Yamanaka’s method provided a way to get such primitive cells without destroying embryos.

In the words of the Nobel committee: “The discoveries of Gurdon and Yamanaka have shown that specialized cells can turn back the developmental clock under certain circumstances. These discoveries have also provided new tools for scientists around the world and led to remarkable progress in many areas of medicine.”

As previously mentioned on this blog, Japanese scientists reported using Yamanaka’s approach to convert skin cells from mice into eggs that produced baby mice.

The 79-year-old Gurdon has served as a professor of cell biology at Cambridge University’s Magdalene College. Currently, he works at the Gurdon Institute in Cambridge, which he founded.

Yamanaka is 50 years old and previously worked at the Gladstone Institute in San Francisco and Nara Institute of Science and Technology in Japan. Currently, Yamanaka is at Kyoto University but retains his affiliation with the Gladstone Institute. Yamanaka is the first Japanese scientist to win the Nobel medicine award since 1987.

The work of Gurdon and Yamanaka earned them a Lasker award for basic research in 2009. Reprogramming cells has also been used in basic research in order to model certain genetic diseases in a culture dish. Cellular reprogramming allows scientists to create particular kinds of tissues with the exact abnormality they want to study. For example, scientists can make lung tissue afflicted with the mutations that cause cystic fibrosis, or brain tissue with Huntington’s disease. By reprogramming cells from patients that have a genetic disease, they can create new tissue with the same genetic flaws, and study it in the lab. Such a strategy can provide new insights into the roots of the problem.

In addition, that approach allows them to screen drugs in the lab for possible new medicines.

It is worth mentioning that Yamanaka’s motivation for developing iPSCs was a moral one. You see, he loved stem cell research, but did not want to destroy embryos. As told in a story published in the New York Times:

Inspiration can appear in unexpected places. Dr. Shinya Yamanaka found it while looking through a microscope at a friend’s fertility clinic. … [H]e looked down the microscope at one of the human embryos stored at the clinic. The glimpse changed his scientific career. “When I saw the embryo, I suddenly realized there was such a small difference between it and my daughters,” said Dr. Yamanaka. … “I thought, we can’t keep destroying embryos for our research. There must be another way.”

Dr. Yamanaka’s training taught him that there is no essential difference between a human embryo and a human adult. Differences in size, location, degree of dependence, and extent of development are not morally significant when it comes to the essential nature of an entity. The embryo is smaller than an adult, but so what? Are smaller people less human? Are the less developed less human? Or course not; they are less adult, but not less human. Does the degree of dependence determine your humanity? Then children and the aged are less human as are people after surgery. Surely this is absurd. Location? Now that’s even more ridiculous. There are the only differences between a human embryo and a human adult and they are purely accidental rather than essential differences.

It is amazing what a moral conviction can do. And note that Yamanaka is not a Christian. Therefore, you cannot say that he is a Christian fundamentalist pro-lifer. His is a purely scientific conclusion.

However, if I may opine on this – Why is human life valuable in the first place? It seems to me that people are valuable because they are a unique creation of God who loves them and values them. Therefore, they are inherently valuable and we should treat each life as precious. The conclusion that the embryo is not essentially different from a human adult is a scientific conclusion, but the conclusion that human life is valuable in the first place, is a religious conclusion.