The science and technology of adult stem cells are running streets ahead of embryonic stem cells. But are the scientific establishment and the mainstream press hushing that up? Dr. Mae-Wan Ho and Prof. Joe Cummins review recent advances that make embryonic stem cells research unethical and unnecessary. Adult stem cells isolated from different tissues are developmentally as flexible as embryonic stem cells. Adult stem cells have been used successfully to repair damaged heart, and to treat a variety of disorders from auto-immune disease to cancer. Furthermore, they can be multiplied for many generations in the laboratory, and established cell lines obtained.
The British science journal, Nature, did a recent series of ‘insight’ articles on stem cells. In the introduction, senior editor Natalie DeWitt refers to the excitement fuelled by "the controversial evidence that adult stem cells have a much higher degree of developmental potential than was previously imagined".
Almost none of the articles address adult stem cells directly, and where they are mentioned, the reader is given the impression that adult stem cells are simply not as good as embryonic stem cells. In the final article on ‘ethical and social considerations’ the author Anne McLaren from the Wellcome/CRC Institute of Cambridge, deplores attempts made to "hype adult stem cells at the expense of ES cells" [1]. And while admitting that ES cells from somatic cell nuclear transfer, SCNT (so-called therapeutic human cloning) "may never prove economic for routine clinical treatment", "they could nonetheless make an immensely valuable contribution to medical research".
McLaren mentioned "a point of view" that SCNT presents no ethical problems, "because the entity to which it gives rise is not an embryo as defined as the product of fusion between sperm and egg, but an artefact, possessing no moral significance". The process did result in Dolly the sheep, but, "Is Dolly perhaps not a sheep, but merely an ovine cyborg?" She asked. So, by a clever escape clause, the bio-ethicist made the moral problem disappear in front of our very eyes.
By the same token, we should not object to reproductive human cloning, for such cloned humans are merely ‘cyborgs’. Nor should we object to making cyborgs that serve real humans in any way we please. (Although it would be problematic to define who the real humans are.) Clearly that could not have been her intention. Her intention was to support embryonic stem cell research.
"Let a thousand stem cell lines bloom" she urged, "but let them bloom in full view of all, so that they can be subject to scientific and ethical review, freely available for research and one day, perhaps, for treating diseases." She has thus pre-empted public opposition to isolating embryonic stem cells from human embryos created by SCNT, and to research done on those cells.
Ironically, the company acknowledged for financial support of the series had a full page advertisement at the beginning of the series, where it describes itself as a company that "develops off-the-shelf cellular products based on the human mesenchymal stem cell (hMSC)". MSC is the best-characterised stem cell to-date, adult or embryonic. Contrary to the claims of the Nature ‘insight’, the scientific establishment has been hyping embryonic stem cells at the expense of adult stem cells, and has already been caught doing so [2].
An article published in Science last year showed that mice cloned from embryonic stem cells by nuclear transfer suffered many genetic defects due to the genetic instability of the embryonic stem cells [3]. According to the Washington Post, a key phrase referring to the genetic instability of the embryonic stem cells that might "limit their use in clinical application", was removed days before the paper appeared in print.
The Statistical Assessment Service (STATS), a non-partisan group dedicated to truth telling in political debates that involve science, found that the mainstream media had given great prominence to the potential of embryonic stem cells while under-reporting or completely ignoring research breakthroughs involving adult stem cells or alternate sources.
The US National Bioethics Advisory Commission (NBAC) recommended to Clinton to support embryonic stem cell research with an important caveat that has generally been ignored. The NBAC recognised that human embryos are destroyed when stem cells are extracted, and it stipulated that this is "justifiable only if no less morally problematic alternatives are available for advancing the research."
It is now clear that such morally less problematic alternatives do exist, in readily available sources of adult stem cells, especially from the patients requiring treatment themselves. We were among those who pointed this out a year ago [4], and numerous subsequent advances have proved our case.
The main protagonists of embryonic stem cell research are the scientists working with those cells. A typical case on why embryonic stem cell research should be supported goes like this [5].
Therefore, the weight of evidence suggests "there are good reasons to want to continue to work with pluripotent stem cells.
In fact, all those arguments have been overtaken by events. There are many excellent peer-reviewed publications showing that adult stem cells may be just as developmentally flexible as embryonic stem cells, that these stem cells show much greater promise in repairing damaged tissues and treatment of other diseases, and that they can give rise to established cell lines, if needed. Furthermore, unlike stem cells isolated from the embryo, they do not carry the same risks of cancer or uncontrollable growth after transplant, and they can be isolated from patients requiring treatment, thus avoiding all problems of immune rejection and the need for immune suppressive drugs that carry their own risks.
The developmental potential of adult stem cells has been documented in numerous publications. For example, bone marrow cells enriched for hematopoietic stem cells (HSC) can differentiate into mature liver cells in the liver of rodents [6, 7] and humans [8, 9]. Mouse bone marrow cells can generate skeletal muscle cells in the body [10, 11], and skeletal muscle cells can give bone marrow cells [12]. Bone marrow could be reconstituted from cultured brain [13], and glial and neurons cells were obtained from bone marrow [14,15].
More recently, a ‘pluripotent neural stem cell’ was isolated from adult mouse brain, where it constitutes 1 in 300 cells of the brain [16]. It not only gave rise to all types of cells in the brain, but when co-cultured with muscle cell line, developed into muscle cells. These neural stem cells can be grown indefinitely in culture.
Researchers have also provided definitive proof that one single adult stem cell from bone marrow can reconstitute the bone marrow of a mouse destroyed by irradiation, as well as develop into practically all the tissues of the body (Box 1). Established cell lines have been obtained from one type of bone marrow stem cells.
Another source of readily available adult stem cells turns out to be skin (Box 2). Skin stem cells can make neurons, glia, smooth muscle and fat cells. Again, cell lines have been established that kept their pluripotency for at least one year.
In summary, adult stem cells are readily available from bone marrow and probably also from skin. These cells are developmentally just as flexible as embryonic stem cells; there appears to be no developmental boundaries that they cannot cross. Furthermore, established cell lines already exist.
Embryonic stem cells are promoted on ground that they are developmentally more flexible than adult stem cells. But too much flexibility may not be desirable. Transplant of embryonic cells into the brains of Parkinson’s patients turned into an irredeemable nightmare because the cells grew uncontrollably [17]. Embryonic stem cells also show genetic instability [3] and carry considerable risks of cancer [4, 18]. When injected under the skin of certain mice, they grow into teratomas, tumors consisting of a jumble of tissue types, from gut to skin to teeth, and the same happens when injected into the brain.
According to a new report [19], mouse embryonic stem cells injected into the brain of adult rats developed into neurons. The rats had previously had their dopamine-producing neurons damaged and showed a characteristic tendency to move in circles toward the damaged side of the brain, mimicking Parkinson’s disease.
Actually, six of the 25 rats injected showed no evidence that the mouse embryonic stem cells had survived. Five died with teratomas in the brain. The 14 remaining showed a modest 40% behavioural improvement over untreated controls, and were found to have mouse stem-cell derived neurons in their brain.
Injected adult stem cells are generally different, they grow into other tissues only when appropriate growth factors are applied, or on other external cues. Indeed, adult stem cells already have a record of relatively safe, effective therapy.
We reported on the use of the patient’s own bone marrow cells to mend damaged heart in a previous issue of ISIS News [20]. A current paper in Nature [21] reviews evidence suggesting that there is continuous cell renewal in adult heart, and that cardiac stem cells can be coaxed into repairing damaged heart. The patient’s bone marrow stem cells have also been successfully used to treat juvenile chronic arthritis, severe systemic lupus erythematosus, an auto-immune disorder with multiple organ dysfunction and Crohn’s disease (Box 3).
Bone marrow transplant from a daughter provided ‘killer cells’ that were tolerated by her mother, thereby ridding the mother of an otherwise untreatable cancer (Box 4).
It is clear that human embryonic stem cell research is both unethical and unnecessary, and should not be supported, least of all with public finance. An indication of the promise of adult stem cells relative to embryonic stem cells is that private finance for the former is outstripping the latter two to one (Box 5).
Our tax money should not be spent to indulge the whims of scientists who cannot see beyond their own self-centred narrow interest, and who are totally insensitive to the moral concerns of the rest of humanity.
Box 1
Single bone marrow stem cell differentiate into blood, liver, lung, gut, skin, bone, cartilage, fat, muscle...
Bone marrow is turning out to be a rich source of readily available adult stem cells. Not all cells in the bone marrow are stem cells. There may also be different kinds of stem cells. Those that normally differentiate into blood cells, hematopoietic stem cells (HSCs), constitute about 1 in 105 cells. The definitive proof that such HSCs can regenerate all the blood cells and other tissues is to purify them, and show that one single cell can multiply and differentiate into all those tissues.
Researchers in Yale Univeristy, New York University and John Hopkins School of Medicine have shown just that.
Bone marrow cells from male mice were purified to produce a population enriched for HSCs. These cells were marked with a membrane bound dye (PKH26) that give a bright colour when illuminated, and injected into female mice that had been irradiated with gamma rays to destroy all their bone marrow cells. Two days after transplant, the PKH26-marked donor cells that still appeared bright – an indication that they had not divided - were recovered by a cell sorter from the recipient bone marrow. By diluting the suspension of the cells, another thirty irradiated females were injected with a single recovered PKH26-labelled cell. These secondary transplant recipients were for followed for 11 months.
In a previous experiment, the researchers found that the cells that homed in to the bone marrow, but not those that homed in to the spleen of the first recipient mouse were capable of repopulating the bone marrow of the second transplant recipient. As a control in the present study, 100 or 1000 purified cells from the male donor were transplanted directly into female recipients without using the homing procedure. None of the control animals survived. On the contrary, 5 of the 30 irradiated female mice that received a single donor cell that had homed in to the bone marrow survived and showed long-term repopulation of the bone marrow and other tissues.
The five long-term survivors were killed at 11 months and cells from each of their bone marrow were plated out and used for further serial transplant. The marrow from 4 of the 5 survivors had between 77.5% and 97.5% donor male-derived colonies. One million cells from each of the five survivors were transplanted into 4 further irradiated females. Four months later, the groups that had received transplants from three of the survivors had 38 to 77% male cells. This represents strong evidence for HSC self-renewal.
The researchers also found that the HSCs that homed to the bone marrow were enriched for specific cell surface markers CD34 and SCA-1.
When they analysed the epithelial tissues from the five mice that had been transplanted with a single homed cell, they found that the single stem cell had descendants in the stomach, small and large intestine, liver, lung and skin.
Significant percentages of donor cells were present in all the tissues examined except kidney. The highest percentage, 18.7% occurred in the lung.
Another research team, from the Stem Cell Institute, Department of Medicine, and Cancer Center, University of Minnesota Medical School, Minneapolis in the United States isolated a different population of stem cells from the bone marrow of human subjects. These were mesenchymal stem cells (MSC). Here, too, a single stem cell was found to differentiate into bone and cartilage cells, skeletal muscle cells, fat cells, bone marrow stroma (ground substance) and endothelial cells (internal linings) of the internal organs. Furthermore, the stem cells can be expanded extensively by means of cell culture.
Bone marrow was collected from 30 healthy human donors (ages 2 to 50 years) following informed consent. These were cultured on fibronectin with epidemal growth factor and platelet-derived growth factor BB and 2% or less foetal calf serum. They found that 1/106 bone marrow mononuclear cells gave rise to clusters of small cells that stuck together. The cell-doubling time was 48 to 72 hours. The cells have been expanded in culture for more than 60 cell doublings. The MCPs (mesodermal progenitor cells arising from the MSC) are found to be distinct from the hematopoietic stem cells (HSCs) in their cell surface proteins. By marking the cells with a retroviral vector carrying green flourescent protein, the researchers showed that single MPCs can differentiate into all the cell types identified.
Their initial hypothesis was that mesodermal progenitors are present in the marrow, but differ from hematopoietic progenitors. So they selected cells that do not express hematopoietic markers (CD45 and GlyA). These constituted 0.1% to 0.5% of bone marrow mononuclear cells. And 0.02% to 0.08% of these CD45-GlyA- cells gave rise to the clusters of sticky cells. These cells were expanded in culture, and found not to express the following markers: CD10, CD31, CD34, CD36, CD38, CED50, CD62E, CD106, CD117, HIP12, fibroblast surface antigen (1B10), HLA-DR, class I HLA, CD45, Tie or Tek. They expressed low levels of beta 2-microglobulin, CD44, CDw90, KDR, and FLTI, and high levels of CD13 and CD59b.
The cell morphology and cell-surface markers remained unchanged for more than 40 cell doublings. Cultures have been established capable of proliferating beyond 30 cell doublings and differentiating to all mesodermal cell types from greater than 85% of donors, ages 2 to 50 years, and MPCs have been expanded for more than 50 cell doublings in at least 10 donors.
The conditions of growth were critical in determining whether the cells remained stem cells or differentiate into one or another cell type. By manipulating the media and culture conditions, different developmental fates were elicited. They could be made to differentiate into bone forming cells, cartilage forming cells, fat cells, skeletal muscle cells and endothelial cells. The researchers have not found differentiation of MCPs into hematopoietic cells, but further studies are going on to test whether additional feeders and cytokine combinations might induce the transition.
To prove that a single cell can differentiate into all cell types, more than 2000 cells recovered from established cultures were plated as single cells in each well of 96-well plates. None of them grew. But when plated at 10 cells per well, progeny of 0.2% of wells could be expanded to more than 107 cells. Thus, MPCs themselves, or accessory cells may secrete growth factors, cytokines (locally acting hormones affecting cell behaviour and differentiation) or extracellular matrix components required for their growth.
Another proof that a single stem cell gave rise to all the tissues was done by marking the cells with a retroviral vector carrying the green flourescent protein gene, and demonstrating that the marked cell differentiated into all the cell types.
Sources:
Box 2
Skin stem cells make neurons, glia, muscle, fat..
Scientists in McGill University, Canada, isolated stem cells from mice skin. The skin was dissociated and cultured with epidermal growth factor and fibroblast growth factor. Cells that floated as small spheres were isolated and transferred to fresh culture. After three to four weeks, the small spheres grew into larger ones. At that point the spheres were dissociated to give single cells. Each cell then grew into a new sphere.
From 8 cm2 of skin containing some 12 million cells, 8000 spheres of cells were obtained in 10 days, each sphere containing 5 to 30 cells. Each of these cells gave rise to half a million to 2-3 million cells. These doubled every two to three days, and have been cultured for at least one year without losing pluripotency.
These cells have the potential to differentiate into neurons, glia, smooth muscle and fat cells.
A preliminary experiment suggested that similar adult stem cells could be isolated from human skin.
Source: Toma HG, Akhavan M, Fernandes KJL, Barnabe-Heider F, Sakikot A, Kaplan DR, and Miller ID. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nature Cell Biol 2001, 3, 778-84.
Box 3
Bone marrow from patients provide treatment for autoimmune diseases
Physicians in the Netherlands (Utrecht and Leiden) treated four children, aged 6 to 11 with juvenile chronic arthritis (JCA), who did not respond to the usual treatments available. Bone marrow was taken from the patients one month before chemotherapy and irradiation depleted the T (thymus) cells, and the patients’ own bone marrow were transplanted back.
The children were followed for 6 to 18 months afterwards when they were maintained free of immune-suppressive drugs. There was rapid reconstitution of the immune system in 3 out of the four children. All showed marked decrease in joint swelling, pain and morning stiffness, as well as improvements in other indicators. Researchers in North Western University, Chicago, meanwhile, had been selecting patients with severe systemic lupus erythematosus for treatment with bone marrow transplants since 1996. These patients did not respond to the usual treatment with cyclosphosphamide (an immune suppressive drug).
Nine patients underwent drug treatment to mobilise their stem cells, two of them were excluded because of infection. The remaining seven underwent the chemotherapy to knock out their immune cells before being transplanted back with their own hematopoietic cells. They were followed up between 12 to 40 months (median 25 mos).
All were free from lupus since; and their renal, cardiac, pulmonary and blood markers remained normal.
More recently, a patient with Crohn’s disease, an autoimmune disorder affecting the digestive system, was successfully treated with a similar procedure.
Sources:
Box 4
Daughter’s stem cells rid mother of cancer
Cells of the foetus are known to persist in mothers for many years. Mothers might therefore be tolerant to cells from their offspring. A 52-year-old Asian woman suffering from carcinoma of the thymus received 1010 white blood cells from her 32-old daughter. The cells were collected after the daughter was given granulocyte macrophage colony stimulating factor for 5 days to boost her white blood cells. The cells were incubated with interleukin-2 (IL-2) for 3h and transfused over 1h without purification before they were transfused into the patient. No immune suppressive drugs were given, and they were not needed.
Within three days of transfusion, the patient stopped coughing and her appetite returned. Her clinical status continued to improve thereafter. By day 210, she had regained her lost weight and resumed a normal active lifestyle. Her tumour had regressed.
The rapid response was attributed to the activity of the natural killer (NK) cells, and to the action of IL-2, which led to further killer cells that cleared the cancer. Tumour markers have been shown to be sharply reduced within one week after the transfusion.
Polymerase Chain Reaction (PCR) studies showed that about 1/100 000 of the patient’s cells were from her daughter before transfusion. Four days after transfusion, this increased to about 1/1000, then decreased gradually to 1:100 000 by day 70 and remained at this level at day 330.
Source: Tokita K, Terasaki P, Maruya E and Saji H. tumour regression following stem cell infusion from daughter to microchimeric mother. Lancet 2001, 358, 2047-48.
Box 5
Private venture capital tells the tale
The venture investment market is voting for adult stem-cell research. There are 30 companies doing stem-cell research. About half of them are involved primarily in developing treatments from stem cells. And only two companies, Geron Corporation and BresaGen, do significant work with embryonic cells. Geron, widely held as the leading embryonic stem cell company, attracted $50 million in investment over the past year, while BresaGen, which works with both embryonic and adult stem cells, received more than $15 million. By contrast, 13 companies working with adult stem cells attracted over $100 million.
Source: "Adult cells do it better" by Scott Gottlieb, American Spectator, http://www.amspec.org/AmericanSpectatorArticles/Gottlieb2.htm
Article first published 11/02/02
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