Category Archives: New York Stem Cells

With more than 50 years of experience studying blood-forming stem cells called hematopoietic stem cells, scientists have developed sufficient understanding to actually use them as a therapy. Currently, no other type of stem cell, adult, fetal or embryonic, has attained such status. Hematopoietic stem cell transplants are now routinely used to treat patients with cancers and other disorders of the blood and immune systems. Recently, researchers have observed in animal studies that hematopoietic stem cells appear to be able to form other kinds of cells, such as muscle, blood vessels, and bone. If this can be applied to human cells, it may eventually be possible to use hematopoietic stem cells to replace a wider array of cells and tissues than once thought.

Despite the vast experience with hematopoietic stem cells, scientists face major roadblocks in expanding their use beyond the replacement of blood and immune cells. First, hematopoietic stem cells are unable to proliferate (replicate themselves) and differentiate (become specialized to other cell types) in vitro (in the test tube or culture dish). Second, scientists do not yet have an accurate method to distinguish stem cells from other cells recovered from the blood or bone marrow. Until scientists overcome these technical barriers, they believe it is unlikely that hematopoietic stem cells will be applied as cell replacement therapy in diseases such as diabetes, Parkinson's Disease, spinal cord injury, and many others.

Blood cells are responsible for constant maintenance and immune protection of every cell type of the body. This relentless and brutal work requires that blood cells, along with skin cells, have the greatest powers of self-renewal of any adult tissue.

The stem cells that form blood and immune cells are known as hematopoietic stem cells (HSCs). They are ultimately responsible for the constant renewal of bloodthe production of billions of new blood cells each day. Physicians and basic researchers have known and capitalized on this fact for more than 50 years in treating many diseases. The first evidence and definition of blood-forming stem cells came from studies of people exposed to lethal doses of radiation in 1945.

Basic research soon followed. After duplicating radiation sickness in mice, scientists found they could rescue the mice from death with bone marrow transplants from healthy donor animals. In the early 1960s, Till and McCulloch began analyzing the bone marrow to find out which components were responsible for regenerating blood [56]. They defined what remain the two hallmarks of an HSC: it can renew itself and it can produce cells that give rise to all the different types of blood cells (see Chapter 4. The Adult Stem Cell).

A hematopoietic stem cell is a cell isolated from the blood or bone marrow that can renew itself, can differentiate to a variety of specialized cells, can mobilize out of the bone marrow into circulating blood, and can undergo programmed cell death, called apoptosisa process by which cells that are detrimental or unneeded self-destruct.

A major thrust of basic HSC research since the 1960s has been identifying and characterizing these stem cells. Because HSCs look and behave in culture like ordinary white blood cells, this has been a difficult challenge and this makes them difficult to identify by morphology (size and shape). Even today, scientists must rely on cell surface proteins, which serve, only roughly, as markers of white blood cells.

Identifying and characterizing properties of HSCs began with studies in mice, which laid the groundwork for human studies. The challenge is formidable as about 1 in every 10,000 to 15,000 bone marrow cells is thought to be a stem cell. In the blood stream the proportion falls to 1 in 100,000 blood cells. To this end, scientists began to develop tests for proving the self-renewal and the plasticity of HSCs.

The "gold standard" for proving that a cell derived from mouse bone marrow is indeed an HSC is still based on the same proof described above and used in mice many years ago. That is, the cells are injected into a mouse that has received a dose of irradiation sufficient to kill its own blood-producing cells. If the mouse recovers and all types of blood cells reappear (bearing a genetic marker from the donor animal), the transplanted cells are deemed to have included stem cells.

These studies have revealed that there appear to be two kinds of HSCs. If bone marrow cells from the transplanted mouse can, in turn, be transplanted to another lethally irradiated mouse and restore its hematopoietic system over some months, they are considered to be long-term stem cells that are capable of self-renewal. Other cells from bone marrow can immediately regenerate all the different types of blood cells, but under normal circumstances cannot renew themselves over the long term, and these are referred to as short-term progenitor or precursor cells. Progenitor or precursor cells are relatively immature cells that are precursors to a fully differentiated cell of the same tissue type. They are capable of proliferating, but they have a limited capacity to differentiate into more than one cell type as HSCs do. For example, a blood progenitor cell may only be able to make a red blood cell (see Figure 5.1. Hematopoietic and Stromal Stem Cell Differentiation ).

Figure 5.1. Hematopoietic and Stromal Stem Cell Differentiation.

( 2001 Terese Winslow, Lydia Kibiuk)

Harrison et al. write that short-term blood-progenitor cells in a mouse may restore hematopoiesis for three to four months [36]. The longevity of short-term stem cells for humans is not firmly established. A true stem cell, capable of self-renewal, must be able to renew itself for the entire lifespan of an organism. It is these long-term replicating HSCs that are most important for developing HSC-based cell therapies. Unfortunately, to date, researchers cannot distinguish the long-term from the short-term cells when they are removed from the bloodstream or bone marrow.

The central problem of the assays used to identify long-term stem cells and short-term progenitor cells is that they are difficult, expensive, and time-consuming and cannot be done in humans. A few assays are now available that test cells in culture for their ability to form primitive and long-lasting colonies of cells, but these tests are not accepted as proof that a cell is a long-term stem cell. Some genetically altered mice can receive transplanted human HSCs to test the cells' self-renewal and hematopoietic capabilities during the life of a mouse, but the relevance of this test for the cells in humanswho may live for decadesis open to question.

The difficulty of HSC assays has contributed to two mutually confounding research problems: definitively identifying the HSC and getting it to proliferate, or increase its numbers, in a culture dish. More rapid research progress on characterizing and using HSCs would be possible if they could be readily grown in the laboratory. Conversely, progress in identifying growth conditions suitable for HSCs and getting the cells to multiply would move more quickly if scientists could reliably and readily identify true HSCs.

HSCs have an identity problem. First, the ones with long-term replicating ability are rare. Second, there are multiple types of stem cells. And, third, the stem cells look like many other blood or bone marrow cells. So how do researchers find the desired cell populations? The most common approach is through markers that appear on the surface of cells. (For a more detailed discussion, see Appendix E.i. Markers: How Do Researchers Use Them to Identify Stem Cells?) These are useful, but not perfect tools for the research laboratory.

In 1988, in an effort to develop a reliable means of identifying these cells, Irving Weissman and his collaborators focused attention on a set of protein markers on the surface of mouse blood cells that were associated with increased likelihood that the cell was a long-term HSC [50]. Four years later, the laboratory proposed a comparable set of markers for the human stem cell [3]. Weissman proposes the markers shown in Table 5.1 as the closest markers for mouse and human HSCs [62].

* Only one of a family of CD59 markers has thus far been evaluated.** Lin- cells lack 13 to 14 different mature blood-lineage markers.

Such cell markers can be tagged with monoclonal antibodies bearing a fluorescent label and culled out of bone marrow with fluorescence-activated cell sorting (FACS).

The groups of cells thus sorted by surface markers are heterogeneous and include some cells that are true, long-term self-renewing stem cells, some shorter-term progenitors, and some non-stem cells. Weissman's group showed that as few as five genetically tagged cells, injected along with larger doses of stem cells into lethally irradiated mice, could establish themselves and produce marked donor cells in all blood cell lineages for the lifetime of the mouse. A single tagged cell could produce all lineages for as many as seven weeks, and 30 purified cells were sufficient to rescue mice and fully repopulate the bone marrow without extra doses of backup cells to rescue the mice [49]. Despite these efforts, researchers remain divided on the most consistently expressed set of HSC markers [27, 32]. Connie Eaves of the University of British Columbia says none of the markers are tied to unique stem cell functions or truly define the stem cell [14]. "Almost every marker I am aware of has been shown to be fickle," she says.

More recently, Diane Krause and her colleagues at Yale University, New York University, and Johns Hopkins University, used a new technique to home in on a single cell capable of reconstituting all blood cell lineages of an irradiated mouse [27]. After marking bone marrow cells from donor male mice with a nontoxic dye, they injected the cells into female recipient mice that had been given a lethal dose of radiation. Over the next two days, some of the injected cells migrated, or homed, to the bone marrow of the recipients and did not divide; when transplanted into a second set of irradiated female mice, they eventually proved to be a concentrated pool of self-renewing stem cells. The cells also reconstituted blood production. The scientists estimate that their technique concentrated the long-term stem cells 500 to 1,000- fold compared with bone marrow.

The classic source of hematopoietic stem cells (HSCs) is bone marrow. For more than 40 years, doctors performed bone marrow transplants by anesthetizing the stem cell donor, puncturing a bonetypically a hipboneand drawing out the bone marrow cells with a syringe. About 1 in every 100,000 cells in the marrow is a long-term, blood-forming stem cell; other cells present include stromal cells, stromal stem cells, blood progenitor cells, and mature and maturing white and red blood cells.

As a source of HSCs for medical treatments, bone marrow retrieval directly from bone is quickly fading into history. For clinical transplantation of human HSCs, doctors now prefer to harvest donor cells from peripheral, circulating blood. It has been known for decades that a small number of stem and progenitor cells circulate in the bloodstream, but in the past 10 years, researchers have found that they can coax the cells to migrate from marrow to blood in greater numbers by injecting the donor with a cytokine, such as granulocyte-colony stimulating factor (GCSF). The donor is injected with GCSF a few days before the cell harvest. To collect the cells, doctors insert an intravenous tube into the donor's vein and pass his blood through a filtering system that pulls out CD34+ white blood cells and returns the red blood cells to the donor. Of the cells collected, just 5 to 20 percent will be true HSCs. Thus, when medical researchers commonly refer to peripherally harvested "stem cells," this is something of a misnomer. As is true for bone marrow, the CD34+ cells are a mixture of stem cells, progenitors, and white blood cells of various degrees of maturity.

In the past three years, the majority of autologous (where the donor and recipient are the same person) and allogeneic (where the donor and recipient are different individuals) "bone marrow" transplants have actually been white blood cells drawn from peripheral circulation, not bone marrow. Richard Childs, an intramural investigator at the NIH, says peripheral harvest of cells is easier on the donorwith minimal pain, no anesthesia, and no hospital staybut also yields better cells for transplants [6]. Childs points to evidence that patients receiving peripherally harvested cells have higher survival rates than bone marrow recipients do. The peripherally harvested cells contain twice as many HSCs as stem cells taken from bone marrow and engraft more quickly. This means patients may recover white blood cells, platelets, and their immune and clotting protection several days faster than they would with a bone marrow graft. Scientists at Stanford report that highly purified, mobilized peripheral cells that have CD34+ and Thy-1+ surface markers engraft swiftly and without complication in breast cancer patients receiving an autologous transplant of the cells after intensive chemotherapy [41].

In the late 1980s and early 1990s, physicians began to recognize that blood from the human umbilical cord and placenta was a rich source of HSCs. This tissue supports the developing fetus during pregnancy, is delivered along with the baby, and, is usually discarded. Since the first successful umbilical cord blood transplants in children with Fanconi anemia, the collection and therapeutic use of these cells has grown quickly. The New York Blood Center's Placental Blood Program, supported by NIH, is the largest U.S. public umbilical cord blood bank and now has 13,000 donations available for transplantation into small patients who need HSCs. Since it began collecting umbilical cord blood in 1992, the center has provided thousands of cord blood units to patients. Umbilical cord blood recipientstypically childrenhave now lived in excess of eight years, relying on the HSCs from an umbilical cord blood transplant [31, 57].

There is a substantial amount of research being conducted on umbilical cord blood to search for ways to expand the number of HSCs and compare and contrast the biological properties of cord blood with adult bone marrow stem cells. There have been suggestions that umbilical cord blood contains stem cells that have the capability of developing cells of multiple germ layers (multipotent) or even all germ layers, e.g., endoderm, ectoderm, and mesoderm (pluripotent). To date, there is no published scientific evidence to support this claim. While umbilical cord blood represents a valuable resource for HSCs, research data have not conclusively shown qualitative differences in the differentiated cells produced between this source of HSCs and peripheral blood and bone marrow.

An important source of HSCs in research, but not in clinical use, is the developing blood-producing tissues of fetal animals. Hematopoietic cells appear early in the development of all vertebrates. Most extensively studied in the mouse, HSC production sweeps through the developing embryo and fetus in waves. Beginning at about day 7 in the life of the mouse embryo, the earliest hematopoietic activity is indicated by the appearance of blood islands in the yolk sac (see Appendix A. Early Development). The point is disputed, but some scientists contend that yolk sac blood production is transient and will generate some blood cells for the embryo, but probably not the bulk of the HSCs for the adult animal [12, 26, 44]. According to this proposed scenario, most stem cells that will be found in the adult bone marrow and circulation are derived from cells that appear slightly later and in a different location. This other wave of hematopoietic stem cell production occurs in the AGMthe region where the aorta, gonads, and fetal kidney (mesonephros) begin to develop. The cells that give rise to the HSCs in the AGM may also give rise to endothelial cells that line blood vessels. [13]. These HSCs arise at around days 10 to 11 in the mouse embryo (weeks 4 to 6 in human gestation), divide, and within a couple of days, migrate to the liver [11]. The HSCs in the liver continue to divide and migrate, spreading to the spleen, thymus, andnear the time of birthto the bone marrow.

Whereas an increasing body of fetal HSC research is emerging from mice and other animals, there is much less information about human fetal and embryonic HSCs. Scientists in Europe, including Coulombel, Peault, and colleagues, first described hematopoietic precursors in human embryos only a few years ago [20, 53]. Most recently, Gallacher and others reported finding HSCs circulating in the blood of 12- to 18-week aborted human fetuses [16, 28, 54] that was rich in HSCs. These circulating cells had different markers than did cells from fetal liver, fetal bone marrow, or umbilical cord blood.

In 1985, it was shown that it is possible to obtain precursors to many different blood cells from mouse embryonic stem cells [9]. Perkins was able to obtain all the major lineages of progenitor cells from mouse embryoid bodies, even without adding hematopoietic growth factors [45].

Mouse embryonic stem cells in culture, given the right growth factors, can generate most, if not all, the different blood cell types [19], but no one has yet achieved the "gold standard" of proof that they can produce long-term HSCs from these sourcesnamely by obtaining cells that can be transplanted into lethally irradiated mice to reconstitute long-term hematopoiesis [32].

The picture for human embryonic stem and germ cells is even less clear. Scientists from James Thomson's laboratory reported in 1999 that they were able to direct human embryonic stem cellswhich can now be cultured in the labto produce blood progenitor cells [23]. Israeli scientists reported that they had induced human ES cells to produce hematopoietic cells, as evidenced by their production of a blood protein, gamma-globin [21]. Cell lines derived from human embryonic germ cells (cultured cells derived originally from cells in the embryo that would ultimately give rise to eggs or sperm) that are cultured under certain conditions will produce CD34+ cells [47]. The blood-producing cells derived from human ES and embryonic germ (EG) cells have not been rigorously tested for long-term self-renewal or the ability to give rise to all the different blood cells.

As sketchy as data may be on the hematopoietic powers of human ES and EG cells, blood experts are intrigued by their clinical potential and their potential to answer basic questions on renewal and differentiation of HSCs [19]. Connie Eaves, who has made comparisons of HSCs from fetal liver, cord blood, and adult bone marrow, expects cells derived from embryonic tissues to have some interesting traits. She says actively dividing blood-producing cells from ES cell cultureif they are like other dividing cellswill not themselves engraft or rescue hematopoiesis in an animal whose bone marrow has been destroyed. However, they may play a critical role in developing an abundant supply of HSCs grown in the lab. Indications are that the dividing cells will also more readily lend themselves to gene manipulations than do adult HSCs. Eaves anticipates that HSCs derived from early embryo sources will be developmentally more "plastic" than later HSCs, and more capable of self-renewal [14].

Scientists in the laboratory and clinic are beginning to measure the differences among HSCs from different sources. In general, they find that HSCs taken from tissues at earlier developmental stages have a greater ability to self-replicate, show different homing and surface characteristics, and are less likely to be rejected by the immune systemmaking them potentially more useful for therapeutic transplantation.

When do HSCs move from the early locations in the developing fetus to their adult "home" in the bone marrow? European scientists have found that the relative number of CD34+ cells in the collections of cord blood declined with gestational age, but expression of cell-adhesion molecules on these cells increased.

The authors believe these changes reflect preparations for the cells to relocatefrom homing in fetal liver to homing in bone marrow [52].

The point is controversial, but a paper by Chen et al. provides evidence that at least in some strains of mice, HSCs from old mice are less able to repopulate bone marrow after transplantation than are cells from young adult mice [5]. Cells from fetal mice were 50 to 100 percent better at repopulating marrow than were cells from young adult mice were. The specific potential for repopulating marrow appears to be strain-specific, but the scientists found this potential declined with age for both strains. Other scientists find no decreases or sometimes increases in numbers of HSCs with age [51]. Because of the difficulty in identifying a long-term stem cell, it remains difficult to quantify changes in numbers of HSCs as a person ages.

A practical and important difference between HSCs collected from adult human donors and from umbilical cord blood is simply quantitative. Doctors are rarely able to extract more than a few million HSCs from a placenta and umbilical cordtoo few to use in a transplant for an adult, who would ideally get 7 to 10 million CD34+ cells per kilogram body weight, but often adequate for a transplant for a child [33, 48].

Leonard Zon says that HSCs from cord blood are less likely to cause a transplantation complication called graft-versus-host disease, in which white blood cells from a donor attack tissues of the recipient [65]. In a recent review of umbilical cord blood transplantation, Laughlin cites evidence that cord blood causes less graft-versus-host disease [31]. Laughlin writes that it is yet to be determined whether umbilical cord blood HSCs are, in fact, longer lived in a transplant recipient.

In lab and mouse-model tests comparing CD34+ cells from human cord with CD34+ cells derived from adult bone marrow, researchers found cord blood had greater proliferation capacity [24]. White blood cells from cord blood engrafted better in a mouse model, which was genetically altered to tolerate the human cells, than did their adult counterparts.

In addition to being far easier to collect, peripherally harvested white blood cells have other advantages over bone marrow. Cutler and Antin's review says that peripherally harvested cells engraft more quickly, but are more likely to cause graft-versus-host disease [8]. Prospecting for the most receptive HSCs for gene therapy, Orlic and colleagues found that mouse HSCs mobilized with cytokines were more likely to take up genes from a viral vector than were non-mobilized bone marrow HSCs [43].

As stated earlier, an HSC in the bone marrow has four actions in its repertoire: 1) it can renew itself, 2) it can differentiate, 3) it can mobilize out of the bone marrow into circulation (or the reverse), or 4) it can undergo programmed cell death, or apoptosis. Understanding the how, when, where, which, and why of this simple repertoire will allow researchers to manipulate and use HSCs for tissue and organ repair.

Scientists have had a tough time trying to growor even maintaintrue stem cells in culture. This is an important goal because cultures of HSCs that could maintain their characteristic properties of self-renewal and lack of differentiation could provide an unlimited source of cells for therapeutic transplantation and study. When bone marrow or blood cells are observed in culture, one often observes large increases in the number of cells. This usually reflects an increase in differentiation of cells to progenitor cells that can give rise to different lineages of blood cells but cannot renew themselves. True stem cells divide and replace themselves slowly in adult bone marrow.

New tools for gene-expression analysis will now allow scientists to study developmental changes in telomerase activity and telomeres. Telomeres are regions of DNA found at the end of chromosomes that are extended by the enzyme telomerase. Telomerase activity is necessary for cells to proliferate and activity decreases with age leading to shortened telomeres. Scientists hypothesize that declines in stem cell renewal will be associated with declines in telomere length and telomerase activity. Telomerase activity in hematopoietic cells is associated with self-renewal potential [40].

Because self-renewal divisions are rare, hard to induce in culture, and difficult to prove, scientists do not have a definitive answer to the burning question: what putsor perhaps keepsHSCs in a self-renewal division mode? HSCs injected into an anemic patient or mouseor one whose HSCs have otherwise been suppressed or killedwill home to the bone marrow and undergo active division to both replenish all the different types of blood cells and yield additional self-renewing HSCs. But exactly how this happens remains a mystery that scientists are struggling to solve by manipulating cultures of HSCs in the laboratory.

Two recent examples of progress in the culturing studies of mouse HSCs are by Ema and coworkers and Audet and colleagues [2, 15]. Ema et al. found that two cytokinesstem cell factor and thrombo-poietinefficiently induced an unequal first cell division in which one daughter cell gave rise to repopulating cells with self-renewal potential. Audet et al. found that activation of the signaling molecule gp130 is critical to survival and proliferation of mouse HSCs in culture.

Work with specific cytokines and signaling molecules builds on several earlier studies demonstrating modest increases in the numbers of stem cells that could be induced briefly in culture. For example, Van Zant and colleagues used continuous-perfusion culture and bioreactors in an attempt to boost human HSC numbers in single cord blood samples incubated for one to two weeks [58]. They obtained a 20-fold increase in "long-term culture initiating cells."

More clues on how to increase numbers of stem cells may come from looking at other animals and various developmental stages. During early developmental stagesin the fetal liver, for exampleHSCs may undergo more active cell division to increase their numbers, but later in life, they divide far less often [30, 42]. Culturing HSCs from 10- and 11-day-old mouse embryos, Elaine Dzierzak at Erasmus University in the Netherlands finds she can get a 15-fold increase in HSCs within the first 2 or 3 days after she removes the AGM from the embryos [38]. Dzierzak recognizes that this is dramatically different from anything seen with adult stem cells and suggests it is a difference with practical importance. She suspects that the increase is not so much a response to what is going on in the culture but rather, it represents the developmental momentum of this specific embryonic tissue. That is, it is the inevitable consequence of divisions that were cued by that specific embryonic microenvironment. After five days, the number of HSCs plateaus and can be maintained for up to a month. Dzierzak says that the key to understanding how adult-derived HSCs can be expanded and manipulated for clinical purposes may very well be found by defining the cellular composition and complex molecular signals in the AGM region during development [13].

In another approach, Lemischka and coworkers have been able to maintain mouse HSCs for four to seven weeks when they are grown on a clonal line of cells (AFT024) derived from the stroma, the other major cellular constituent of bone marrow [39]. No one knows which specific factors secreted by the stromal cells maintain the stem cells. He says ongoing gene cloning is rapidly zeroing in on novel molecules from the stromal cells that may "talk" to the stem cells and persuade them to remain stem cellsthat is, continue to divide and not differentiate.

If stromal factors provide the key to stem cell self-renewal, research on maintaining stromal cells may be an important prerequisite. In 1999, researchers at Osiris Therapeutics and Johns Hopkins University reported culturing and expanding the numbers of mesenchymal stem cells, which produce the stromal environment [46]. Whereas cultured HSCs rush to differentiate and fail to retain primitive, self-renewing cells, the mesenchymal stem cells could be increased in numbers and still retained their powers to generate the full repertoire of descendant lineages.

Producing differentiated white and red blood cells is the real work of HSCs and progenitor cells. M.C. MacKey calculates that in the course of producing a mature, circulating blood cell, the original hematopoietic stem cell will undergo between 17 and 19.5 divisions, "giving a net amplification of between ~170,000 and ~720,000" [35].

Through a series of careful studies of cultured cellsoften cells with mutations found in leukemia patients or cells that have been genetically alteredinvestigators have discovered many key growth factors and cytokines that induce progenitor cells to make different types of blood cells. These factors interact with one another in complex ways to create a system of exquisite genetic control and coordination of blood cell production.

Scientists know that much of the time, HSCs live in intimate connection with the stroma of bone marrow in adults (see Chapter 4. The Adult Stem Cell). But HSCs may also be found in the spleen, in peripheral blood circulation, and other tissues. Connection to the interstices of bone marrow is important to both the engraftment of transplanted cells and to the maintenance of stem cells as a self-renewing population. Connection to stroma is also important to the orderly proliferation, differentiation, and maturation of blood cells [63].

Weissman says HSCs appear to make brief forays out of the marrow into tissues, then duck back into marrow [62]. At this time, scientists do not understand why or how HSCs leave bone marrow or return to it [59]. Scientists find that HSCs that have been mobilized into peripheral circulation are mostly non-dividing cells [64]. They report that adhesion molecules on the stroma, play a role in mobilization, in attachment to the stroma, and in transmitting signals that regulate HSC self-renewal and progenitor differentiation [61].

The number of blood cells in the bone marrow and blood is regulated by genetic and molecular mechanisms. How do hematopoietic stem cells know when to stop proliferating? Apoptosis is the process of programmed cell death that leads cells to self-destruct when they are unneeded or detrimental. If there are too few HSCs in the body, more cells divide and boost the numbers. If excess stem cells were injected into an animal, they simply wouldn't divide or would undergo apoptosis and be eliminated [62]. Excess numbers of stem cells in an HSC transplant actually seem to improve the likelihood and speed of engraftment, though there seems to be no rigorous identification of a mechanism for this empirical observation.

The particular signals that trigger apoptosis in HSCs are as yet unknown. One possible signal for apoptosis might be the absence of life-sustaining signals from bone marrow stroma. Michael Wang and others found that when they used antibodies to disrupt the adhesion of HSCs to the stroma via VLA-4/VCAM-1, the cells were predisposed to apoptosis [61].

Understanding the forces at play in HSC apoptosis is important to maintaining or increasing their numbers in culture. For example, without growth factors, supplied in the medium or through serum or other feeder layers of cells, HSCs undergo apoptosis. Domen and Weissman found that stem cells need to get two growth factor signals to continue life and avoid apoptosis: one via a protein called BCL-2, the other from steel factor, which, by itself, induces HSCs to produce progenitor cells but not to self-renew [10].

Among the first clinical uses of HSCs were the treatment of cancers of the bloodleukemia and lymphoma, which result from the uncontrolled proliferation of white blood cells. In these applications, the patient's own cancerous hematopoietic cells were destroyed via radiation or chemotherapy, then replaced with a bone marrow transplant, or, as is done now, with a transplant of HSCs collected from the peripheral circulation of a matched donor. A matched donor is typically a sister or brother of the patient who has inherited similar human leukocyte antigens (HLAs) on the surface of their cells. Cancers of the blood include acute lymphoblastic leukemia, acute myeloblastic leukemia, chronic myelogenous leukemia (CML), Hodgkin's disease, multiple myeloma, and non-Hodgkin's lymphoma.

Thomas and Clift describe the history of treatment for chronic myeloid leukemia as it moved from largely ineffective chemotherapy to modestly successful use of a cytokine, interferon, to bone marrow trans-plantsfirst in identical twins, then in HLA-matched siblings [55]. Although there was significant risk of patient death soon after the transplant either from infection or from graft-versus-host disease, for the first time, many patients survived this immediate challenge and had survival times measured in years or even decades, rather than months. The authors write, "In the space of 20 years, marrow transplantation has contributed to the transformation of [chronic myelogenous leukemia] CML from a fatal disease to one that is frequently curable. At the same time, experience acquired in this setting has improved our understanding of many transplant-related problems. It is now clear that morbidity and mortality are not inevitable consequences of allogeneic transplantation, [and] that an allogeneic effect can add to the anti-leukemic power of conditioning regimens"

In a recent development, CML researchers have taken their knowledge of hematopoietic regulation one step farther. On May 10, 2001, the Food and Drug Administration approved Gleevec (imatinib mesylate), a new, rationally designed oral drug for treatment of CML. The new drug specifically targets a mutant protein, produced in CML cancer cells, that sabotages the cell signals controlling orderly division of progenitor cells. By silencing this protein, the new drug turns off cancerous overproduction of white blood cells, so doctors do not have to resort to bone marrow transplantation. At this time, it is unknown whether the new drug will provide sustained remission or will prolong life for CML patients.

Another use of allogeneic bone marrow transplants is in the treatment of hereditary blood disorders, such as different types of inherited anemia (failure to produce blood cells), and inborn errors of metabolism (genetic disorders characterized by defects in key enzymes need to produce essential body components or degrade chemical byproducts). The blood disorders include aplastic anemia, beta-thalassemia, Blackfan-Diamond syndrome, globoid cell leukodystrophy, sickle-cell anemia, severe combined immunodeficiency, X-linked lymphoproliferative syndrome, and Wiskott-Aldrich syndrome. Inborn errors of metabolism that are treated with bone marrow transplants include: Hunter's syndrome, Hurler's syndrome, Lesch Nyhan syndrome, and osteopetrosis. Because bone marrow transplantation has carried a significant risk of death, this is usually a treatment of last resort for otherwise fatal diseases.

Chemotherapy aimed at rapidly dividing cancer cells inevitably hits another targetrapidly dividing hematopoietic cells. Doctors may give cancer patients an autologous stem cell transplant to replace the cells destroyed by chemotherapy. They do this by mobilizing HSCs and collecting them from peripheral blood. The cells are stored while the patient undergoes intensive chemotherapy or radiotherapy to destroy the cancer cells. Once the drugs have washed out of a patient's body, the patient receives a transfusion of his or her stored HSCs. Because patients get their own cells back, there is no chance of immune mismatch or graft-versus-host disease. One problem with the use of autologous HSC transplants in cancer therapy has been that cancer cells are sometimes inadvertently collected and reinfused back into the patient along with the stem cells. One team of investigators finds that they can prevent reintroducing cancer cells by purifying the cells and preserving only the cells that are CD34+, Thy-1+[41].

One of the most exciting new uses of HSC transplantation puts the cells to work attacking otherwise untreatable tumors. A group of researchers in NIH's intramural research program recently described this approach to treating metastatic kidney cancer [7]. Just under half of the 38 patients treated so far have had their tumors reduced. The research protocol is now expanding to treatment of other solid tumors that resist standard therapy, including cancer of the lung, prostate, ovary, colon, esophagus, liver, and pancreas.

This experimental treatment relies on an allogeneic stem cell transplant from an HLA-matched sibling whose HSCs are collected peripherally. The patient's own immune system is suppressed, but not totally destroyed. The donor's cells are transfused into the patient, and for the next three months, doctors closely monitor the patient's immune cells, using DNA fingerprinting to follow the engraftment of the donor's cells and regrowth of the patient's own blood cells. They must also judiciously suppress the patient's immune system as needed to deter his/her T cells from attacking the graft and to reduce graft-versus-host disease.

A study by Joshi et al. shows that umbilical cord blood and peripherally harvested human HSCs show antitumor activity in the test tube against leukemia cells and breast cancer cells [22]. Grafted into a mouse model that tolerates human cells, HSCs attack human leukemia and breast cancer cells. Although untreated cord blood lacks natural killer (NK) lymphocytes capable of killing tumor cells, researchers have found that at least in the test tube and in mice, they can greatly enhance the activity and numbers of these cells with cytokines IL-15 [22, 34].

Substantial basic and limited clinical research exploring the experimental uses of HSCs for other diseases is underway. Among the primary applications are autoimmune diseases, such as diabetes, rheumatoid arthritis, and system lupus erythematosis. Here, the body's immune system turns to destroying body tissues. Experimental approaches similar to those applied above for cancer therapies are being conducted to see if the immune system can be reconstituted or reprogrammed. More detailed discussion on this application is provided in Chapter 6. Autoimmune Diseases and the Promise of Stem Cell-Based Therapies. The use of HSCs as a means to deliver genes to repair damaged cells is another application being explored. The use of HSCs for gene therapies is discussed in detail in Chapter 11. Use of Genetically Modified Stem Cells in Experimental Gene Therapies.

A few recent reports indicate that scientists have been able to induce bone marrow or HSCs to differentiate into other types of tissue, such as brain, muscle, and liver cells. These concepts and the experimental evidence supporting this concept are discussed in Chapter 4. The Adult Stem Cell.

Research in a mouse model indicates that cells from grafts of bone marrow or selected HSCs may home to damaged skeletal and cardiac muscle or liver and regenerate those tissues [4, 29]. One recent advance has been in the study of muscular dystrophy, a genetic disease that occurs in young people and leads to progressive weakness of the skeletal muscles. Bittner and colleagues used mdx mice, a genetically modified mouse with muscle cell defects similar to those in human muscular dystrophy. Bone marrow from non-mdx male mice was transplanted into female mdx mice with chronic muscle damage; after 70 days, researchers found that nuclei from the males had taken up residence in skeletal and cardiac muscle cells.

Lagasse and colleagues' demonstration of liver repair by purified HSCs is a similarly encouraging sign that HSCs may have the potential to integrate into and grow in some non-blood tissues. These scientists lethally irradiated female mice that had an unusual genetic liver disease that could be halted with a drug. The mice were given transplants of genetically marked, purified HSCs from male mice that did not have the liver disease. The transplants were given a chance to engraft for a couple of months while the mice were on the liver-protective drug. The drug was then removed, launching deterioration of the liverand a test to see whether cells from the transplant would be recruited and rescue the liver. The scientists found that transplants of as few as 50 cells led to abundant growth of marked, donor-derived liver cells in the female mice.

Recently, Krause has shown in mice that a single selected donor hematopoietic stem cell could do more than just repopulate the marrow and hematopoietic system of the recipient [27]. These investigators also found epithelial cells derived from the donors in the lungs, gut, and skin of the recipient mice. This suggests that HSCs may have grown in the other tissues in response to infection or damage from the irradiation the mice received.

In humans, observations of male liver cells in female patients who have received bone marrow grafts from males, and in male patients who have received liver transplants from female donors, also suggest the possibility that some cells in bone marrow have the capacity to integrate into the liver and form hepatocytes [1].

Clinical investigators share the same fundamental problem as basic investigatorslimited ability to grow and expand the numbers of human HSCs. Clinicians repeatedly see that larger numbers of cells in stem cell grafts have a better chance of survival in a patient than do smaller numbers of cells. The limited number of cells available from a placenta and umbilical cord blood transplant currently means that cord blood banks are useful to pediatric but not adult patients. Investigators believe that the main cause of failure of HSCs to engraft is host-versus-graft disease, and larger grafts permit at least some donor cells to escape initial waves of attack from a patient's residual or suppressed immune system [6]. Ability to expand numbers of human HSCs in vivo or in vitro would clearly be an enormous boost to all current and future medical uses of HSC transplantation.

Once stem cells and their progeny can be multiplied in culture, gene therapists and blood experts could combine their talents to grow limitless quantities of "universal donor" stem cells, as well as progenitors and specific types of red and white blood cells. If the cells were engineered to be free of markers that provoke rejection, these could be transfused to any recipient to treat any of the diseases that are now addressed with marrow, peripheral, cord, or other transfused blood. If gene therapy and studies of the plasticity of HSCs succeed, the cells could also be grown to repair other tissues and treat non-blood-related disorders [32].

Several research groups in the United States, Canada, and abroad have been striving to find the key factor or factors for boosting HSC production. Typical approaches include comparing genes expressed in primitive HSCs versus progenitor cells; comparing genes in actively dividing fetal HSCs versus adult HSCs; genetic screening of hematopoietically mutated zebrafish; studying dysregulated genes in cancerous hematopoietic cells; analyzing stromal or feeder-layer factors that appear to boost HSC division; and analyzing factors promoting homing and attachment to the stroma. Promising candidate factors have been tried singly and in combination, and researchers claim they can now increase the number of long-term stem cells 20-fold, albeit briefly, in culture.

The specific assays researchers use to prove that their expanded cells are stem cells vary, which makes it difficult to compare the claims of different research groups. To date, there is only a modest ability to expand true, long-term, self-renewing human HSCs. Numbers of progenitor cells are, however, more readily increased. Kobari et al., for example, can increase progenitor cells for granulocytes and macrophages 278-fold in culture [25].

Some investigators are now evaluating whether these comparatively modest increases in HSCs are clinically useful. At this time, the increases in cell numbers are not sustainable over periods beyond a few months, and the yield is far too low for mass production. In addition, the cells produced are often not rigorously characterized. A host of other questions remainfrom how well the multiplied cells can be altered for gene therapy to their potential longevity, immunogenicity, ability to home correctly, and susceptibility to cancerous transformation. Glimm et al. [17] highlight some of these problems, for example, with their confirmation that human stem cells lose their ability to repopulate the bone marrow as they enter and progress through the cell cyclelike mouse stem cells that have been stimulated to divide lose their transplantability [18]. Observations on the inverse relationship between progenitor cell division rate and longevity in strains of mice raise an additional concern that culture tricks or selection of cells that expand rapidly may doom the cells to a short life.

Pragmatically, some scientists say it may not be necessary to be able to induce the true, long-term HSC to divide in the lab. If they can manipulate progenitors and coax them into division on command, gene uptake, and differentiation into key blood cells and other tissues, that may be sufficient to accomplish clinical goals. It might be sufficient to boost HSCs or subpopulations of hematopoietic cells within the body by chemically prodding the bone marrow to supply the as-yet-elusive factors to rejuvenate cell division.

Currently, the risks of bone marrow transplantsgraft rejection, host-versus-graft disease, and infection during the period before HSCs have engrafted and resumed full blood cell productionrestrict their use to patients with serious or fatal illnesses. Allogeneic grafts must come from donors with a close HLA match to the patient (see Chapter 6. Autoimmune Diseases and the Promise of Stem Cell-Based Therapies). If doctors could precisely manipulate immune reactions and protect patients from pathogens before their transplants begin to function, HSC transplants could be extended to less ill patients and patients for whom the HLA match was not as close as it must now be. Physicians might use transplants with greater impunity in gene therapy, autoimmune disease, HIV/AIDS treatment, and the preconditioning of patients to accept a major organ transplant.

Scientists are zeroing in on subpopulations of T cells that may cause or suppress potentially lethal host-versus-graft rejection and graft-versus-host disease in allogeneic-transplant recipients. T cells in a graft are a two-edged sword. They fight infections and help the graft become established, but they also can cause graft-versus-host disease. Identifying subpopulations of T cells responsible for deleterious and beneficial effectsin the graft, but also in residual cells surviving or returning in the hostcould allow clinicians to make grafts safer and to ratchet up graft-versus-tumor effects [48]. Understanding the presentation of antigens to the immune system and the immune system's healthy and unhealthy responses to these antigens and maturation and programmed cell death of T cells is crucial.

The approach taken by investigators at Stanfordpurifying peripheral bloodmay also help eliminate the cells causing graft-versus-host disease. Transplants in mouse models support the idea that purified HSCs, cleansed of mature lymphocytes, engraft readily and avoid graft-versus-host disease [60].

Knowledge of the key cellular actors in autoimmune disease, immune grafting, and graft rejection could also permit scientists to design gentler "minitransplants." Rather than obliterating and replacing the patient's entire hematopoietic system, they could replace just the faulty components with a selection of cells custom tailored to the patient's needs. Clinicians are currently experimenting with deletion of T cells from transplants in some diseases, for example, thereby reducing graft-versus-host disease.

Researchers are also experimenting with the possibility of knocking down the patient's immune systembut not knocking it out. A blow that is sublethal to the patient's hematopoietic cells given before an allogeneic transplant can be enough to give the graft a chance to take up residence in the bone marrow. The cells replace some or all of the patient's original stem cells, often making their blood a mix of donor and original cells. For some patients, this mix of cells will be enough to accomplish treatment objectives but without subjecting them to the vicious side effects and infection hazards of the most powerful treatments used for total destruction of their hematopoietic systems [37].

At some point in embryonic development, all cells are plastic, or developmentally flexible enough to grow into a variety of different tissues. Exactly what is it about the cell or the embryonic environment that instructs cells to grow into one organ and not another?

Could there be embryological underpinnings to the apparent plasticity of adult cells? Researchers have suggested that a lot of the tissues that are showing plasticity are adjacent to one another after gastrulation in the sheet of mesodermal tissue that will go on to form bloodmuscle, blood vessels, kidney, mesenchyme, and notochord. Plasticity may reflect derivation from the mesoderm, rather than being a fixed trait of hematopoietic cells. One lab is now studying the adjacency of embryonic cells and how the developing embryo makes the decision to make one tissue instead of anotherand whether the decision is reversible [65].

In vivo studies of the plasticity of bone marrow or purified stem cells injected into mice are in their infancy. Even if follow-up studies confirm and more precisely characterize and quantify plasticity potential of HSCs in mice, there is no guarantee that it will occur or can be induced in humans.

Grounded in half a century of research, the study of hematopoietic stem cells is one of the most exciting and rapidly advancing disciplines in biomedicine today. Breakthrough discoveries in both the laboratory and clinic have sharply expanded the use and supply of life-saving stem cells. Yet even more promising applications are on the horizon and scientists' current inability to grow HSCs outside the body could delay or thwart progress with these new therapies. New treatments include graft-versus-tumor therapy for currently incurable cancers, autologous transplants for autoimmune diseases, and gene therapy and tissue repair for a host of other problems. The techniques, cells, and knowledge that researchers have now are inadequate to realize the full promise of HSC-based therapy.

Key issues for tapping the potential of hematopoietic stem cells will be finding ways to safely and efficiently expand the numbers of transplantable human HSCs in vitro or in vivo. It will also be important to gain a better understanding of the fundamentals of how immune cells workin fighting infections, in causing transplant rejection, and in graft-versus-host disease as well as master the basics of HSC differentiation. Concomitant advances in gene therapy techniques and the understanding of cellular plasticity could make HSCs one of the most powerful tools for healing.

Chapter 4|Table of Contents|Chapter 6

Historical content: June 17, 2001

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5. Hematopoietic Stem Cells | stemcells.nih.gov

Hans Clevers pioneered lab-built mini-organs that can serve as models ofdisease

A basic biologist at heart, Clevers says he never expected his findings tobenefit patients.

By her 50th birthday, Els van der Heijden felt sicker than ever. Born with the hereditarydisorder cystic fibrosis (CF), she had managed to work around her illness, finishingcollege and landing a challenging job in consulting. But Van der Heijden, who lives in asmall Dutch town, says she always felt a dark cloud hanging over myhead. When she began feeling exhausted and easily out of breath in 2015, shethought it was the beginning of the end.

Then she read a newspaper article about a child with CF named Fabian whose life had beensaved after scientists grew a mini-organ from a tissue sample snippedfrom his colon, one organ that CF affects. Doctors had used the mini-organ to testivacaftor (Kalydeco), a drug so expensive that Dutch insurers refuse to cover it withoutevidence that it will help an individual CF patient. No such data existed for Fabian,whose CF was caused by an extremely rare mutation. But his minigut responded toivacaftor, and he improved within hours of taking it. His insurance eventually agreed topay for the drug.

Van der Heijden's doctor arranged to have a minigut made for her as well; itresponded to a drug marketed as Orkambi that combines ivacaftor and another compound,lumacaftor. Within weeks after she began taking that combination, I had anenormous amount of energy, she says. For the first time ever, I feltlike my body was functioning like it should.

The life-altering test was developed in the lab of Hans Clevers, director of the HubrechtInstitute here. More than a decade ago, Clevers identified a type of mother cell in thegut that can give birth to all other intestinal cells. With the right nutrition, histeam coaxed such stem cells to grow into a 3D, pencil tip-sized version of the gut fromwhich it came. The minigut was functionally similar to the intestine and replete withall its major cell typesan organoid.

That was the start of a revolution. Clevers and others have since grown organoids frommany other organs, including the stomach, pancreas, brain, and liver. Easy tomanipulate, organoids are clarifying how tissues develop and repair injury. But perhapsmost exciting, many researchers say, is their ability to model diseases in new ways.Researchers are creating organoids from tumor cells to mimic cancers and introducingspecific mutations into organoids made from healthy tissue to study how cancer arises.And as Clevers's lab has shown, organoids can help predict how an individual willrespond to a drugmaking personalized medicine a reality. It is highlylikely that organoids will revolutionize therapy of many severe diseases, saysRudolf Jaenisch, a stem cell scientist at the Massachusetts Institute of Technology inCambridge.

For Clevers, the bonanza has come as a surprise. A basic biologist at heart, he says henever had real-world applications in mind. I was always driven bycuriosity, he says. For 25 years we published papers with no practicalrelevance for anyone on this planet.

ON A BRIGHT JULY MORNING at the Hubrecht Institute, Clevers listenspatiently to presentations during a weekly lab meeting. One postdoc presents data on herefforts to develop an organoid model for small-cell lung cancer; another reportsprogress on culturing hormone-secreting organoids from human gut tissue. Whenever theirresearch questions strike him as uninspired, Clevers urges them to be more ambitious:Why don't you pursue something you don't know? he asks.

Hans is capable of raising questions that are not contaminated by the anticipatedanswer, says Edward Nieuwenhuis, chairman of pediatrics at University MedicalCenter Utrecht (UMCU) and a good friend. He has a better nose than most forsniffing around and finding interesting stuff, says Ronald Plasterk, whoco-directed the Hubrecht lab with Clevers from 2002 to 2007 and is now the DutchMinister of the Interior and Kingdom Relations. That approach has earned Clevers manyawards. In June, for example, he was inducted into the Orden Pour le Mrite, anelite German order with just 80 members worldwide.

Clevers began his career studying immune cells as a postdoc at the Dana-Farber CancerInstitute in Boston. He landed his first job at UMCU's clinical immunologydepartment in 1989, where he quickly became department head. Most of the work wasclinical, such as leukemia diagnostics and blood work for transplants. But myresearch interests were always much more basic than the environment that I wasin, he says.

In early work, he identified a key molecule, T cell-specific transcription factor 1(TCF-1), that signals the immune cells known as T lymphocytes to proliferate. Later hefound that TCF-1 is part of the larger Wnt family of signaling molecules that'simportant not only for immune responses, but also for embryonic development and tissuerepair. In 1997, his lab team discovered that mice lacking the gene for one of thosesignals, TCF-4, failed to develop pockets in their intestinal lining called crypts. Soonafter, a study with Bert Vogelstein at Johns Hopkins University in Baltimore, Maryland,showed that TCF-4 also helps initiate human colon cancer. Fascinated, Clevers switchedhis focus from the immune system to the gut.

Inspired by a flurry of research on stem cells at the time, Clevers began hunting forintestinal stem cells. More than 50 years ago, researchers deduced that rodent cryptsproduce many cells that survive only a few days, suggesting some unidentified,longer-lived source for the cells.

After almost a decade of tedious experiments, Clevers's postdoc Nick Barker struckgold in 2007: He discovered that cells carrying a receptor named LGR5 give rise to allcells in mouse intestines and that molecules in the Wnt pathway signal those cells todivide. Barker later found LGR5-positive cells in other organs as well. In some, thecells were always active; in others, such as the liver, they multiplied only whentissues sensed injury.

At the time, culturing stem cells was notoriously hard, but after combing throughprevious lab experiments, another postdoc in Clevers's lab, Toshiro Sato, concocteda mix of growth factors that coaxed the gut stem cells to replicate in a dish. He hopedto see a flat layer of cells. But what emerged in 2009 from a single LGR5-positive cellwas a beautiful structure that surprised and intrigued me, says Sato,now at Keio University in Tokyo: a 3D replica of a gut epithelium. The structureself-organized into crypts and finger-shaped protrusions called villi, and it beganmaking its own biochemicals. A paper about the feat was rejected several times beforebeing published. Clevers recalls: No one wanted to believe it.

Soon, the lab began culturing LGR5-positive cells and growing organoids from the stomach,liver, and other organs. It was an exciting time, and I really felt like we wereon the frontiers of discovery, says another postdoc at the time, Meritxell Huch,now at the Gurdon Institute in Cambridge, U.K. But we certainly didn'tthink we were opening a new field.

CAPTIVATED BY STEM CELLS and their potential to regenerate tissues, otherlabs were starting to make organoids. A few months before Sato's 2009 paper,Akifumi Ootani, a postdoc in Calvin Kuo's group at Stanford University in PaloAlto, California, reported using a different strategy to grow gut organoids. Kuo'smethod starts with tissue fragments rather than individual stem cells and grows them ina gel partly exposed to air instead of submerged in nutrient medium. Around the sametime, Yoshiki Sasai of the RIKEN Center for Developmental Biology in Kobe, Japan,cultured the first brain organoids, starting not with adult stem cells but withembryonic stem cells. Other researchers grew organoids from induced pluripotent stemcells, which resemble embryonic stem cells but are grown from adult cells.

The various methods create different kinds of organoids, each with advantages anddrawbacks. Kuo's organoids contain a mix of cell types, which enablesobservation of higher-order behaviors such as muscle contraction, hesays. Because those organoids include stroma, a scaffold of connective tissue essentialfor tumor growth, they may prove better for studying therapies that target the stroma,such as cancer immunotherapy. Clevers's mix of growth factors grows organoidsconsisting primarily of epithelial cells, so his technique doesn't work for thebrain and other organs with few or no epithelial cells. Nor can his organoids be used totest drugs targeting blood vessels or immune cells because organoids have neither.

Both methods can generate organoids from individual patients, producing a personalizedminigut in just 1 to 3 weeks. (Although Clevers's organoids originate from adultstem cells, isolating those cells isn't necessary; culturing a tissue fragment withthe right nutrients is enough.) The methods are reproducible, and the organoids remaingenetically stable in culture; they can also be stored in freezers for years.

In 2013, Clevers and others founded a nonprofit, Hubrecht Organoid Technology (HUB), tomarket applications. Clevers first proposed using organoids for tissue transplants, saysHUB Managing Director Rob Vries. Studies showed that healthy organoids implanted in micewith diseased colons could repair injury. But we bagged the idea because therewere too many regulatory hurdles and the chance of success was low, Vriessays.

Cystic fibrosis patient Els van der Heijden received a new drug combination basedon organoid tests. Within weeks, I had an enormous amount ofenergy, she says.

The idea of enlisting organoids to treat CF came from Jeffrey Beekman, a researcher atUMCU who studies that disease. All Dutch newborns are screened for CF, and colon biopsysamples are taken from babies who test positive. The tissue is tested to gauge howdysfunctional the defective gene is and then stored. Growing organoids from thosesamples would be relatively simple, argued Beekman, who has since spearheaded theproject.

CF can arise from more than 2000 mutations in one gene, which cripple the ion channelsthat move salt and water through cell membranes. The disease affects all tissues, butthe primary symptom is excess mucus in the lungs and gut, causing chest infections,coughing, difficulty breathing, and digestive problems.

Ivacaftor and the combination drug lumacaftor and ivacaftor, both marketed by VertexPharmaceuticals in Boston, restore the ion channels' function. But the drugsdon't work equally well for everyone, and they have been tested and approved onlyfor people with the most common mutations, together accounting for roughly half of allCF patients. Vertex, which declined to answer questions for this story, has beenreluctant to spend millions on trials in patients with rare mutations because thepotential payoff is small. And with the price tagboth drugs cost between100,000 and 200,000 per year in Europehealth services andinsurance companies have been unwilling to pay for the medicines for people with thoseuntested mutations.

Van der Heijden falls into that category because only two other people in the Netherlandsshare her mutation. But when organoids grown from her gut were exposed to lumacaftor andivacaftor, the organoids swelled like normal gut tissue, a sign that the defectiveprotein was working and that salt and water were flowing through. The result helpedpersuade Vertex to give her the drug through a compassionate-use program, withoutpayment. (Regulatory agencies require her to be monitored in a clinical trial.) Her sideeffects included fatigue, nausea, and diarrhea, but after a few months, it wasas if someone opened the curtain and said, Look, the sun is there, come out andplay, she says. And I did.

In collaboration with Vertex, HUB has tested ivacaftor on organoids grown from CFpatients who had taken part in a clinical trial of that drug. The study confirmed thatorganoids can predict who will respond to the drug.

HUB has also tested ivacaftor on organoids from 50 patients with nine rare mutations. Onthe basis of the results, insurers agreed to pay for the drug in six more Dutchpatients, and Vertex is following up with the first clinical trial of ivacaftor in CFpatients with rare mutations. Meanwhile, HUB is building a biobank, financed by Dutchhealth insurers, containing organoids from all 1500 Dutch CF patients for testing bothexisting drugs and new candidates.

This is the next big thing in CF research, says Eitan Kerem, head ofpediatrics at Hadassah Medical Center in Jerusalem, who is building a similar biobankand has launched a trial in patients with rare mutations. Organoids are especiallyuseful because no great animal models for CF exist, Kerem says; ferrets and pigs aresometimes used, but they are expensive and not available to mostresearchers.

Drug and biotech companies are now striking deals with HUB to explore organoids in otherdiseases. The success with CF suggests that they can model other single-gene disorders,such as -1 antitrypsin deficiency, which causes symptoms primarily in the lungsand liver. Some companies are also testing failed drugs on organoids and comparing theresults with animal and clinical data, hoping to find ways to predict and avoid suchfailures.

CANCER IS ALSO a major target. By growing organoids from tumor samples,researchers can create minitumors and use them to study how cancer develops or to testdrugs. Soon after the minigut paper came out in 2009, David Tuveson, who heads thecancer center at Cold Spring Harbor Laboratory in New York, began prodding Clevers todevelop organoids for pancreatic cancer, which is notoriously hard to treat. Existingcell culture models were not very realistic, Tuveson says, and creating geneticallyengineered mice took up to a year, compared with up to 3 weeks for pancreatic cancerorganoids.

The organoids have already helped clarify new pathways that lead to pancreatic cancer,Tuveson says, and unpublished data suggest that they will help researchers predict whichtreatments will be most effective. He and Clevers are trying to make the organoidsresemble real cancer more closely by adding stroma and immune cells. The Hubrecht lab isalso involved in two trials to assess whether colon cancer organoids grown fromindividual patients can predict drug response.

Charles Sawyers of Memorial Sloan Kettering Cancer Center in New York City is trying tomake prostate cancer organoids, but he says they are finicky. Organoids from primarytumors generally don't grow; those from metastatic tissue sometimes do, but normalcells often outgrow cancer cells. They seem to need a lot of tender love andcare, and there is no method to the madness, says Sawyers, who has succeededwith only 20 patients so far.

Organoids can be used to study how pathogens interact with human tissues. In thislung organoid grown in Hans Clevers's lab, cells colored green are infectedwith respiratory syncytial virus.

But Sawyers discovered that he could easily grow organoids from normal prostatetissueit just works beautifully, he saysand then usegene-editing techniques such as CRISPR to study any cancer mutation he wants. Isthis a tumor suppressor gene? Is this an oncogene? Does it collaborate with geneXY? You can play the kind of games on the scale that you alwayswanted to, he says. As Kuo puts it, We can build cancer from the groundup.

Other cancer researchers want in, too. Tuveson received so many requests for organoidtraining that he began hosting regular workshops at his laboratory. In 2016, the U.S.National Cancer Institute launched a scheme to develop more than 1000 cell culturemodels, including organoids, for researchers around the world to use, together withCancer Research UK in London, the Wellcome Trust Sanger Institute in Hinxton, U.K., andHUB.

Using personalized organoids to treat cancer still faces hurdles. Organoid culture time,which varies by cancer, must be shortened, and the cost, a few thousand dollars perpatient, needs to come down. Also, cancers accumulate genetic mutations as theyprogress, which could mean that an organoid grown from a patient's cancer early onmight not reflect its later state. Nevertheless, from my perspective it'sthe most transformative advance in cancer research that I know of, Tuvesonsays.

If all of that excites Clevers, he rarely shows it. He avoids emotional language whilediscussing his research, preferring instead to describe and explain. Even close friendssometimes find his pragmatism puzzling. He talks about his research like someonetalking about screwing in a screw, Nieuwenhuis says.

Clevers says he gets his high from the satisfaction of finding somethingnovel, regardless of practical applications. Recent experiments, for instance,suggest that when an organ lacks LGR-5-positive cells, differentiated cells may be ableto dedifferentiate and repair tissuesa radical change from theone-way street toward specific identities that stem cells were thought to travel.Some organs may not have a professional stem cell at all, Clevers says,with a hint of wonder. But when asked how he felt when he saw his findings have profoundbenefits for patients such as Fabian and Els van der Heijden, he simply says, Idid not expect that.

Read the original here:
The organoid architect - Science Magazine

There are many claims that stem cells possess anti-ageing properties and other secrets to youth and regeneration. However, there has not been much scientific proof demonstrating these touted abilities.

Dr Paul Lucas, an assistant professor of orthopaedics and pathology from the New York Medical College in the United States, notes that the words stem cells are thrown around far too casually, and that many people assume that they are a single type of cell.

The definition of stem cell is an operational definition.

That is, it describes what the cell can do, and not any particular protein or other marker it can make, he says.

According to him, a stem cell is a cell that can:

Differentiate into at least one phenotype (cell type), and

Has the ability to divide, with at least one daughter cell remaining a stem cell.

Lots of hype, very little biology. I have written several answers on the website Quora that address this.

Pills and creams are not legit.

The skin has a barrier called the stratum corneum that prevents bacteria from getting inside the body.

The stratum corneum will also block stem cells, which are much, much larger than bacteria, in the form of a cream.

Any stem cell will not survive in a pill with no water. And of course, any cell will not survive the hydrochloric acid in the stomach.

So there is no way stem cells in either a pill or a cream can get inside the body.

Even if a stem cell could get inside the body, there is very little data that any stem cell will be anti-ageing its a way to separate people from their money.

There are several reasons stem cells do not counter ageing.

Stem cells are not magic. They are not magic pixie dust you can sprinkle on everything and make it be perfect.

Ageing has many causes. One of them is DNA and cellular damage.

It is thought that the various adult stem cells are the cells of origin of cancer. The data is very solid for at least hepatomas and leukaemias.

That means that stem cells can suffer mutations that alter cellular function degrading it in some cases, and causing it to go haywire and be cancer in others.

Also, how are stem cells to be injected? Into each tissue? Every muscle, organ, tendon, ligament, etc?

Or are the stem cells to be injected into a vein and travel to all parts of the body?

There are two technical problems with this:

Injecting into a vein means that most of the cells are going to be trapped in the lungs before they go out to the rest of the body, as our veins all lead first to our heart, then our lungs.

Blood vessels are sealed tubes. Think pipes.

Just how are the stem cells supposed to exit the pipes?

This is especially true for reversing ageing in the most important organ the brain.

The neural tissue in the brain is separated from the blood vessels by another layer of tissue called the blood-brain barrier.

Even if stem cells got out of the blood vessels in the brain, they are not going to get to the neural tissue, which is the tissue that needs to rejuvenate.

There is no way any injected stem cells are just going to magically replace all the aged cells in the body.

Stem cells are a class of undifferentiated cells that are able to differentiate into specialised cell types. Photo: 123rf.com

Plants are very different from us. No cell from a plant is going to be able to incorporate into our tissues and act like a stem cell.

Many mammalian stem cells particularly mesenchymal stem cells synthesise and secrete several proteins.

Some of these proteins are growth factors in that they cause other cells to divide.

The claim seems to be that plant growth factors will have the same effect on human cells as they do on plant cells.

That is false.

Even some of the skincare people admit this. The following quote is from the website of a US-based skincare company that uses both human and plant stem cells: That said, unlike human stem cells, the growth factors, cytokines and other proteins, which are the products of plant stem cells, do not have the ability to act in the same way in humans, as in plants.

Plant stem cells communicate in a different biochemical language that human cells do not recognise.

First is the source.

ESCs are the inner cell mass of a five to seven-day-old blastocyst, which is formed after the sperm successfully fertilises the egg.

PSCs come either from the tissue of the placenta itself or from the Whartons jelly of the umbilical cord.

Secondly, ESCs are pluripotent, meaning they are able to differentiate into every tissue of the body. They can also form tumours in our body.

PSCs are essentially adult stem cells that have limited proliferation potential, i.e. the cell has a fixed number of times it can divide before it dies. They are multipotent, meaning that they have the ability to form more than one cell type, and do not form tumours.

Probably less costly, but no more effective.

The treatment uses mesenchymal stem cells (MSCs).

The discoverer of MSCs Prof Dr Arnold Caplan says they should be called mesenchymal secreting cells. Notice that he does not consider them stem cells!

MSCs secrete a large number of cytokines that reduce inflammation. It is inflammation that causes pain.

Aspirin, ibuprofen, and naproxen also reduce inflammation.

A stem cell injection with MSCs is essentially putting little aspirin factories at the site of injury.

They reduce the pain, but do little or nothing to regenerate the tissue.

For young athletes, reducing inflammation will allow the bodys healing process to work better, and thus, improve outcome.

For older patients? There is less capacity for healing.

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Are stems cells really the fountain of youth? - Star2.com - Star2.com

UTRECHT, Netherlands Els van der Heijden, who has cystic fibrosis, was finding it ever harder to breathe as her lungs filled with thick, sticky mucus. Despite taking more than a dozen pills and inhalers a day, the 53-year-old had to stop working and scale back doing the thing she loved best, horseback riding.

Doctors saw no sense in trying an expensive new drug because it hasnt been proven to work in people with the rare type of cystic fibrosis that van der Heijden had.

Instead, they scraped a few cells from van der Heijden and used them to grow a mini version of her large intestine in a petri dish. When van der Heijdens mini gut responded to treatment, doctors knew it would help her too.

I really felt, physically, like a different person, van der Heijden said after taking a drug and getting back in the saddle.

This experiment to help people with rare forms of cystic fibrosis in the Netherlands aims to grow mini intestines for every Dutch patient with the disease to figure out, in part, what treatment might work for them. Its an early application of a technique now being worked on in labs all over the world, as researchers learn to grow organs outside of the body for treatment and maybe someday for transplants.

So far, doctors have grown mini guts just the size of a pencil point for 450 of the Netherlands roughly 1,500 cystic fibrosis patients.

The mini guts are small, but they are complete, said Dr. Hans Clevers of the Hubrecht Institute, who pioneered the technique. Except for muscles and blood vessels, the tiny organs have everything you would expect to see in a real gut, only on a really small scale.

These so-called organoids mimic features of full-size organs but dont function the same way. Although many of the tiny replicas are closer to undeveloped organs found in an embryo than adult ones, they are helping scientists unravel how organs mature and providing clues on how certain diseases might be treated.

In Australia, mini kidneys are being grown that could be used to test drugs. Researchers in the US are experimenting with tiny bits of livers that might be used to boost failing organs. At Cambridge University in England, scientists have created hundreds of mini brains to study how neurons form and better understand disorders like autism. During the height of the Zika epidemic last year, mini brains were used to show the virus causes malformed brains in babies.

In the Netherlands, the mini guts are used as a stand-in for cystic fibrosis patients to see if those with rare mutations might benefit from a number of pricey drugs, including Orkambi. Made by Vertex Pharmaceuticals, Orkambi costs about 100,000 euros ($118,000) per patient every year in some parts of Europe and its more than double that in the US, which approved the drug in 2015. Despite being initially rejected by the Dutch government for being too expensive, negotiations with Vertex were reopened in July.

Making a single mini gut and testing whether the patient would benefit from certain drugs costs a couple of thousand euros. The program is paid for by groups including health insurance companies, patient foundations and the government. The idea is to find a possible treatment for patients and avoid putting them on expensive drugs that wouldnt work for them.

About 50 to 60 patients across the Netherlands have been treated after drugs were tested on organoids using their cells, said Dr. Kors van der Ent, a cystic fibrosis specialist at the Wilhelmina Childrens Hospital, who leads the research.

Clevers made a discovery about a decade ago that got researchers on their way. They found pockets of stem cells, which can turn into many types of other cells, in the gut. They then homed in a growing environment in the lab that spurred these cells to reproduce rapidly and develop.

To our surprise, the stem cells started building a mini version of the gut, Clevers recalled.

Cystic fibrosis is caused by mutations in a single gene that produces a protein called CFTR, responsible for balancing the salt content of cells lining the lungs and other organs.

To see if certain drugs might help cystic fibrosis patients, the medicines are given to their custom-made organoids in the lab. If the mini organs puff up, its a sign the cells are now correctly balancing salt and water. That means the drugs are working and could help the patient from whom the mini-gut was made.

Researchers are also using the mini guts to try another approach they hope will someday work in people using a gene editing technique to repair the faulty cystic fibrosis gene in the organoid cells.

Other experiments are underway in the Netherlands and the US to test whether organoids might help pinpoint treatments for cancers involving lungs, ovaries and pancreas.

While the idea sounds promising, some scientists said there are obstacles to using mini organs to study cancer.

Growing a mini cancer tumor, for example, would be far more challenging because scientists have found it difficult to make tumors in the lab that behave like in real life, said Mathew Garnett of the Wellcome Trust Sanger Institute, who has studied cancer in mini organs but is not connected to Clevers research.

Also, growing the cells and testing them must happen faster for cancer patients who might not have much time to live, he said.

Meanwhile, Clevers wants to one day make organs that are not so mini.

My dream would be to be able to custom-make organs, he said, imagining a future where doctors might have a freezer full of livers to choose from when sick patients arrive.

Others said while such a vision is theoretically possible, huge hurdles remain.

There are still enormous challenges in tissue engineering with regards to the size of the structure were able to grow, said Jim Wells, a pediatrics professor at the Cincinnati Childrens Hospital Medical Center. He said the mini organs are far smaller than what would be needed to transplant into people and its unclear if scientists can make a working, life-sized organ in the lab.

There are other limitations to growing miniature organs in a dish, said Madeline Lancaster at Cambridge University.

We can study physical changes and try to generate drugs that could prevent detrimental effects of disease, but we cant look at the complex interplay between organs and the body, she said.

For patients like van der Heijden, who was diagnosed with cystic fibrosis as a toddler, the research has helped her regain her strength. Vertex agreed to supply her with the drug.

It was like somebody opened the curtains and said, Sunshine, here I am, please come out and play.' she said. Its strange to think this is all linked to some of my cells in a lab.

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'Mini-organs' help personalize treatments for cystic fibrosis patients - New York Post

New Jersey Blood Services (NJBS), a division of New York Blood Center (NYBC), is urging the public to donate blood as we head into the Labor Day holiday. New Jersey roads are packed with cars heading to the beach and the mountains. With so many people on vacation, blood donations drop significantly the last week of August into the first ten days of September as parents and students prepare for the start of the school year.

The need for blood never takes a holiday. Blood donations are urgently needed this time of year. Not only do we see fewer blood donations, but fewer blood drives are scheduled during this two week period. Patients in local hospitals still need blood for emergencies and regular treatments, however.

NYBC announced a blood emergency in late June which lasted much of the summer. Raising awareness for this critical Labor Day holiday period will help boost blood donations and rebuild the inventory.

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O negative blood donors are considered universal, and their blood type is needed most readily in trauma situations and emergency departments across the country. Due to its high demand, O negative blood is in short supply and NYBC encourages individuals with this blood type to donate today. Our local blood supply has reached a critically low level, with under a two-day supply of O negative, B negative, and A negative.

To donate blood or for information on how to organize a blood drivePlease call Toll Free: 1-800-933-2566Visit: http://www.nybloodcenter.org/blood

In order to maintain a safe blood supply a seven-day inventory of all types must be continually replenished. Companies, organizations, and community groups are also encouraged to step up to host a blood drive in September to help rebuild the blood supply. Hosting a blood drive is easy and NYBCs staff will help every step of the way.

More About Blood Donations

The entire donation process takes less than an hour and a single donation can be used to save multiple lives. Donors with O-negative blood type, or universal donors, are especially encouraged to donate, as their blood can be used in emergencies. Nearly 2,000 donations are needed each day in New York and New Jersey alone. About one in seven hospital admissions requires a blood transfusion, and with a limited shelf life, supplies must be continually replenished.

If you cannot donate but still wish to participate in bringing crucial blood products to patients in need, please consider hosting a blood drive or volunteering at a local blood drive.

Any company, community organization, place of worship, or individual may host a blood drive. Blood donors receive free mini-medical exams on site including information about their temperature, blood pressure and hematocrit level. Eligible donors include those people at least age 16 (parental consent is required for 16-year-olds), who weigh a minimum of 110 pounds, are in good health and meet all Food & Drug Administration and NY or NJ State Department of Health donor criteria. People age 76 or older may donate if they have a doctors note on file with New York Blood Center or if they bring one on the day of the blood drive.

About New York Blood Center

Now more than 50 years old, New York Blood Center (NYBC) is a nonprofit organization that is one of the largest independent, community-based blood centers in the country. NYBCs mission is to serve the 20 million people in the New York metropolitan area and more broadly, our nation and our world by alleviating human suffering and preserving human life using our medical expertise.

Each year, NYBC provides approximately one million blood products to nearly 200 hospitals in the Northeast. NYBC also provides a wide array of transfusion-related medical services. NYBC is also home to the worlds largest public cord blood bank, which provides stem cells for transplant in many countries, and a renowned research institute, which among other milestones developed the hepatitis B vaccine and innovative blood purification technology.

Website: nybc.org

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Blood Donations needed pre and post Labor Day Holiday! - TAPinto.net

Amelia-Grace Harpham is a 16-year-old with wild, wavy hair and a love of reading. She prefers sci-fi shows like Dr. Who and Star Trek to the usual teen fare. And the rising junior at Hastings HS in Hastings-on-Hudson always says yes to a competitive game of capture the flag.

Its hard to believe that little more than a decade ago, this vibrant teen (who then went by Gracie) was fighting for her life against a rare blood disease that left doctors baffled. In 2004 and 2005, The Post ran several stories about the then-toddlers mysterious disease and generous readers helped raise $85,000 for a bone marrow transplant that saved her life.

Today, the only telltale signs of Amelias fragile past are the scars on her chest from various treatments.

A couple of friends have seen them, she told The Post. People are interested, but they dont know the scope of it.

Hers was a harrowing experience riddled with frightening prognoses and statistics. One doctor predicted she had only a 50 percent chance of living past the age of 29. Her mother, Heather Harpham, has chronicled it all in a memoir, Happiness: The Crooked Little Road to Semi-Ever After (Henry Holt, out now).

When Amelia was born on March 30, 2001, she looked perfectly healthy; but hours after her birth, doctors realized something was malfunctioning in her blood. She had her first transfusion within the first week of her life, and it would be the start of a scary routine that spanned her next three years. Unable to produce sufficient red blood cells, the baby needed transfusions about every three weeks.

After each transfusion, Amelia would look pink and healthy. But that energy would drain like a battery, and she would turn wan and listless until the next one. The family was told that becoming reliant on transfusions could eventually prove lethal.

Doctors never landed on a specific diagnosis or cause, but they were certain of one thing: Amelia would be cured by a bone marrow transplant.

But the procedure on a toddler doesnt come without risks. The ideal situation would involve using stem cells harvested from a newborns umbilical cord rather than an adults bone marrow. A doctor suggested that Heather and her partner (now husband), Brian Morton, have another baby, because a sibling would have a one in four chance of being a match. Afraid of having two tragically ill children, the couple resisted the idea.

But shortly thereafter, Heather discovered she was unexpectedly pregnant. After baby boy Gabriel was born, blood from his umbilical cord was saved and indeed, he was an exact match for his then-2-year-old sisters bone marrow.

Doctors again stressed, however, that such a transplant process, which also involved chemotherapy, could be grueling for the toddler and possibly overwhelm her fragile body. Heather and Brian who lived in Park Slope at the time made the agonizing decision to go ahead with the risky procedure at Duke University Medical Center in Durham, NC. But there was still more bad news: They would need to pay $85,000 not covered by insurance.

Thats when neighbor Kathy Sears jumped in. As covered by The Post at the time, she organized a block party and a raffle with all of the proceeds going toward Amelias medical treatments, Perfect strangers from all over the city turned up.

People came out of the woodwork, asking how they could help, Sears recalled. Little old ladies walking up to me shoving $25 checks into my hand. It was so beautiful.

Heather remembered a man showing up to the block party with an envelope of money collected from his colleagues at the Department of Motor Vehicles.

All told, more than $85,000 was raised, which helped when the tab came to more than first anticipated. According to Heather, money given by Post readers made up the bulk of the donations.

I cannot overestimate what Post readers did. It gave us the sense that all of New York was in our corner. In this odd way, I would wish for anyone to have the experience of being in need, being fragile and being frightened and [then to] be utterly embraced, supported and carried by a community, she said.

Amelia now has no physical restrictions. She visits Duke every other year for checkups, but is more focused on her studies.

I enjoy all of my subjects, but I love English, she said. I would like to be a writer. I keep journals of important stuff thats happened.

She also has a typical sibling relationship with Gabriel, now 14. As their mom explains: Its like, You can give me the remote, I gave you my stem cells.

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How New York Post readers helped save a toddler's life - New York Post

Thats because none of those talents arise from a single gene mutation, or even from an easily identifiable number of genes. Most human traits are nowhere near that simple.

Right now, we know nothing about genetic enhancement, said Hank Greely, director of the Center for Law and the Biosciences at Stanford. Were never going to be able to say, honestly, This embryo looks like a 1550 on the two-part SAT.

Even with an apparently straightforward physical characteristic like height, genetic manipulation would be a tall order. Some scientists estimate height is influenced by as many as 93,000 genetic variations. A recent study identified 697 of them.

A new technique known as Crispr has revolutionized humans ability to edit DNA. See you if you can identify whether a given development has already happened, could eventually happen or is pure fiction.

You might be able to do it with something like eye color, said Robin Lovell-Badge, a professor of genetics and embryology at the Francis Crick Institute in London.

But if people are worried about designer babies, theyre normally thinking of doing special different things than the normal genetic stuff.

The gene-modification process used in the new study also turns out to be somewhat restrictive. After researchers snipped the harmful mutation from the male gene, it copied the healthy sequence from that spot on the female gene.

That was a surprise to the scientists, who had inserted a DNA template into the embryo, expecting the gene to copy that sequence into the snipped spot, as occurs with gene editing in other body cells. But the embryonic genome ignored that template, suggesting that to repair a mutation on one parents gene in an embryo, a healthy DNA sequence from the other parent is required.

If you cant introduce a template, then you cant do anything wild, Dr. Lovell-Badge said. This doesnt really help you make designer babies.

Talents and traits arent the only thing that are genetically complex. So are most physical diseases and psychiatric disorders. The genetic message is not carried in a 140-character tweet it resembles a shelf full of books with chapters, subsections and footnotes.

So embryonic editing is unlikely to prevent most medical problems.

But about 10,000 medical conditions are linked to specific mutations, including Huntingtons disease, cancers caused by BRCA genes, Tay-Sachs disease, cystic fibrosis, sickle cell anemia, and some cases of early-onset Alzheimers. Repairing the responsible mutations in theory could eradicate these diseases from the so-called germline, the genetic material passed from one generation to the next. No future family members would inherit them.

But testing editing approaches on each mutation will require scientists to find the right genetic signpost, often an RNA molecule, to guide the gene-snipping tool.

In the study reported this week, it took 10 tries to find the right RNA, said Juan Carlos Izpisua Belmonte, a co-author and geneticist at the Salk Institute.

Dr. Greely noted that while scientists work to get human embryonic editing ready for clinical trials (currently illegal in the United States and many countries), alternate medical treatments for these diseases might be developed. They may be simpler and cheaper.

How good one technique is depends on how good the alternatives are, and there may be alternatives, he said.

The authors of the new study do not dismiss ethical implications of their work. In fact, Dr. Belmonte served on a committee of the National Academies of Science, Engineering and Medicine that in February endorsed research into gene editing of human embryos, but only to prevent serious diseases and conditions, and as a last resort.

In theory this could lead to the kind of intervention which, of course, Im totally against, said Dr. Belmonte. The possibility of moving forward not to create or prevent disease but rather to perform gene enhancement in humans.

For example, soon we will know more and more about genes that can increase your muscle activity, he said. The hormone EPO, which some athletes have been disciplined for taking, is produced by a gene, so you could in theory engineer yourself to produce more EPO.

That is the kind of genetic engineering that raises alarm.

Allowing any form of human germline modification leaves the way open for all kinds especially when fertility clinics start offering genetic upgrades to those able to afford them, Marcy Darnovsky, executive director of the Center for Genetics and Society, said in a statement. We could all too easily find ourselves in a world where some peoples children are considered biologically superior to the rest of us.

Scientists and ethicists share the concerns about access. Any intervention that goes to the clinic should be for everyone, Dr. Belmonte said. It shouldnt create inequities in society.

Unequal access is, of course, a question that arises with almost any new medical intervention, and already disparities deprive too many people of needed treatments.

But there is a flip side to ethical arguments against embryo editing.

I personally feel we are duty bound to explore what the technology can do in a safe, reliable manner to help people, Dr. Lovell-Badge said. If you have a way to help families not have a diseased child, then it would be unethical not to do it.

Genetic engineering doesnt have to be an all or nothing proposition, some scientists and ethicists say. There is a middle ground to stake out with laws, regulation and oversight.

For example, Dr. Lovell-Badge said, Britain highly regulates pre-implantation genetic diagnosis, in which a couples embryos are screened for certain harmful mutations so that only healthy ones are implanted in the womans womb.

They allow sensible things to be done, and they dont allow non-sensible things, he said. And every single embryo is accounted for. If someone tries to do something they shouldnt have done, they will find out, and the penalties for breaking the law are quite severe.

According to a 2015 article in the journal Nature, a number of countries, including the United States, restrict or ban genetic modification of human embryos.

Other countries, like China, have guidelines but not laws banning or restricting clinical use, the article noted. Chinese researchers have conducted the only previously published gene editing experiments on human embryos, which were much less successful.

In the future, will there be nations that allow fertility clinics to promise babies with genetically engineered perfect pitch or .400 batting averages? Its not impossible. Even now, some clinics in the United States and elsewhere offer unproven stem cell therapies, sometimes with disastrous consequences.

But R. Alta Charo, a bioethicist at University of Wisconsin-Madison, who co-led the national committee on human embryo editing, said historically ethical overreach with reproductive technology has been limited.

Procedures like I.V.F. are arduous and expensive, and many people want children to closely resemble themselves and their partners. They are likely to tinker with genes only if other alternatives are impractical or impossible.

You hear people talking about how this will make us treat children as commodities and make people more intolerant of people with disabilities and lead to eugenics and all that, she said.

While I appreciate the fear, I think we need to realize that with every technology we have had these fears, and they havent been realized.

Read more here:
Gene Editing for 'Designer Babies'? Highly Unlikely, Scientists Say - New York Times

Wednesday, August 2, 2017

The vast majority of genetic mutations that are associated with diseaseoccur at sites in the genome that arent genes. These sequences of DNA dont code for proteins themselves, but provide an additional layer of instructions that determine if and when particular genes are expressed. Researchers are only beginning to understand how the non-coding regions of the genome influence gene expression andmight be disrupted in disease.

Jennifer Phillips-Cremins, assistant professor in the Department of Bioengineering in the University of Pennsylvanias School of Engineering and Applied Science, studies the three-dimensional folding of the genome and the role it plays in brain development. When a stretch of DNA folds, it creates a higher-order structure called a looping interaction, or loop. In doing so , it brings non-coding sites into physicalcontact with their target genes to precisely regulate gene expression in space and time during development.

Phillips-Cremins and lab member Jonathan Beagan have led a new study identifying a new protein that connects loops in embryonic stem cells as they begin to differentiate into types of neurons. Though the study was conducted in mice, these findings inform aspects of human brain development, including how the genetic material folds in the 3-D nucleus and is reconfigured as stem cells become specialized. Better understanding of these mechanisms may be relevant to a wide range of neurodevelopmental disorders.

Cremins lab members Michael Duong, Katelyn Titus, Linda Zhou, Zhendong Cao, Jingjing Ma, Caroline Lachanski and Daniel Gillis also contributed to the study, which was published in the journal Genome Research.

In this paper we create detailed maps of how the genetic material, the DNA, folds in three dimensions inside cells in the brain. We uncover a new class of looping interactions that emerge only when embryonic stem cells turn into neural stem cells in the brain, Phillips-Cremins said. These neural stem-cell-specific loops are important because they connect non-coding regulatory elements to their target genes at a developmental stage when brain-specific gene expression patterns are initially established.

We also discovered that most new neural stem-cell-specific loops arise within a larger framework of pre-existing loops that are established far earlier in development and present in most cell types in the body, Beagan said.

A protein named CTCF is known to be the main connector of loops that are stable throughout development. The researchers discovered that CTCF is sharply reduced in the transition from early development to neural stem cells, leading to a global pruning back of loops that dont matter for the brain and leaving only the stable framework in place.

The Cremins lab creates experimental heat maps of the higher-order structure of the genome. By fixing the DNA such that its 3-D folding patterns are preserved prior to sequencing, two distant parts of the linear sequence will end up in the same string of hybrid DNA and will thus be detected together when the DNA is sequenced.

In this study we integrated maps of protein binding to the DNA with our 3-D genome heat maps. We unexpectedly discovered that the traditional architectural protein CTCF does not connect brain-specific loops, Beagan said. Rather, we found a new protein, Ying Yang 1, or YY1, that is essential for connecting the 3D genome specifically in early stages of brain development. Disruption of this protein has been implicated in brain diseases in early human development.

At this early stage, we can only say YY1 plays an essential role in connecting brain-specific loops at the earliest stages of neurodevelopment. However, brain development and maturation is a complex process and were excited to continue to unravel the organizing principles governing genome folding in fully differentiated neurons in the human brain, Phillips-Cremins said. Because the large majority of disease-associated mutations are located in the non-coding regions of the genome, these results might eventually shed light on the mechanisms underlying the onset and progression of a wide range of neurodevelopmental diseases.

This research was supported by The New York Stem Cell Foundation, the Alfred P. Sloan Foundation, the National Institute of Mental Health through the NIH Director's New Innovator Award (1DP2MH11024701), the National Institutes of Health through a 4D Nucleome Common Fund grant (1U01HL12999801), and a joint National Science Foundation - National Institute of General Medical Sciences grant (1562665). This material is based upon work supported by the National Science Foundation Graduate Research Fellowship grant (DGE-1321851).

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Penn Engineers Identify Protein Implicated in 3-D Epigenetics of Brain Development - Penn Current

New York, Aug 4 (IANS) A new form of gene therapy administered through skin transplants can help improve treatments for Type-2 diabetes and obesity, researchers have claimed.

Using CRISPR, researchers from the University of Chicago edited the skin stem cells from newborn mice which prompted the cells to secrete glucagon-like peptide 1 (GLP1) -- a hormone that stimulates the pancreas to secrete insulin and regulates blood sugar.

The cells when transplanted onto mice showed the grafts increased insulin secretion and reversed weight gain from a high-fat diet, as well as overturned insulin resistance.

"We resolved some technical hurdles and designed a mouse-to-mouse skin transplantation model in animals with intact immune systems," said Xiaoyang Wu, Assistant Professor at the University of Chicago.

"We think this platform has the potential to lead to safe and durable gene therapy, in mice and we hope, someday, in humans, using selected and modified cells from skin," Wu added.

Further, the researchers inserted one mutation, designed to extend the hormone's half-life in the blood stream, and fused the modified gene to an antibody fragment so that GLP-1 would circulate in the blood stream longer.

They also attached an inducible promoter, which enabled them to turn on the gene to make more GLP1, as needed, by exposing it to the antibiotic doxycycline, the researchers said in the paper detailed in the journal Cell Stem Cell.

When the mice were fed minute amounts of doxycycline, they released GLP1 into the blood, which promptly increased blood-insulin levels and reduced blood-glucose levels.

When high-fat diet was combined with doxycycline, the mice secreted GLP1 and gained less weight, suggesting that "cutaneous gene therapy for GLP1 secretion could be practical and clinically relevant".

"We think this can provide a long-term safe option for the treatment of many diseases. It could be used to deliver therapeutic proteins, replacing missing proteins for people with a genetic defect such as hemophilia. Or it could function as a metabolic sink, removing various toxins," Wu said.

--IANS

rt/py/vt

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Gene therapy via skin may treat diabetes, obesity - Economic Times

Altering human heredity? In a first, researchers safely repaired a disease-causing gene in human embryos, targeting a heart defect best known for killing young athletes a big step toward one day preventing a list of inherited diseases.

In a surprising discovery, a research team led by Oregon Health and & Science University reported Wednesday that embryos can help fix themselves if scientists jump-start the process early enough.

It's laboratory research only, nowhere near ready to be tried in a pregnancy. But it suggests that scientists might alter DNA in a way that protects not just one baby from a disease that runs in the family, but his or her offspring as well. And that raises ethical questions.

"I for one believe, and this paper supports the view, that ultimately gene editing of human embryos can be made safe. Then the question truly becomes, if we can do it, should we do it?" said Dr. George Daley, a stem cell scientist and dean of Harvard Medical School. He wasn't involved in the new research and praised it as "quite remarkable."

"This is definitely a leap forward," agreed developmental geneticist Robin Lovell-Badge of Britain's Francis Crick Institute.

Today, couples seeking to avoid passing on a bad gene sometimes have embryos created in fertility clinics so they can discard those that inherit the disease and attempt pregnancy only with healthy ones, if there are any.

Gene editing in theory could rescue diseased embryos. But so-called "germline" changes altering sperm, eggs or embryos are controversial because they would be permanent, passed down to future generations. Critics worry about attempts at "designer babies" instead of just preventing disease, and a few previous attempts at learning to edit embryos, in China, didn't work well and, more importantly, raised safety concerns.

In a series of laboratory experiments reported in the journal Nature, the Oregon researchers tried a different approach.

They targeted a gene mutation that causes a heart-weakening disease, hypertrophic cardiomyopathy, that affects about 1 in 500 people. Inheriting just one copy of the bad gene can cause it.

The team programmed a gene-editing tool, named CRISPR-Cas9, that acts like a pair of molecular scissors to find that mutation a missing piece of genetic material.

Then came the test. Researchers injected sperm from a patient with the heart condition along with those molecular scissors into healthy donated eggs at the same time. The scissors cut the defective DNA in the sperm.

Normally cells will repair a CRISPR-induced cut in DNA by essentially gluing the ends back together. Or scientists can try delivering the missing DNA in a repair package, like a computer's cut-and-paste program.

Instead, the newly forming embryos made their own perfect fix without that outside help, reported Oregon Health & Science University senior researcher Shoukhrat Mitalipov.

We all inherit two copies of each gene, one from dad and one from mom and those embryos just copied the healthy one from the donated egg.

"The embryos are really looking for the blueprint," Mitalipov, who directs OHSU's Center for Embryonic Cell and Gene Therapy, said in an interview. "We're finding embryos will repair themselves if you have another healthy copy."

It worked 72 percent of the time, in 42 out of 58 embryos. Normally a sick parent has a 50-50 chance of passing on the mutation.

Drew Angerer/Getty Images

Previous embryo-editing attempts in China found not every cell was repaired, a safety concern called mosaicism. Beginning the process before fertilization avoided that problem: Until now, "everybody was injecting too late," Mitalipov said.

Nor did intense testing uncover any "off-target" errors, cuts to DNA in the wrong places, reported the team, which also included researchers from the Salk Institute for Biological Studies in California and South Korea's Institute for Basic Science. None of the embryos was allowed to develop beyond eight cells, a standard for laboratory research.

Genetics and ethics experts not involved in the work say it's a critical first step but just one step toward eventually testing the process in pregnancy, something currently prohibited by U.S. policy.

"This is very elegant lab work," but it's moving so fast that society needs to catch up and debate how far it should go, said Johns Hopkins University bioethicist Jeffrey Kahn.

And lots more research is needed to tell if it's really safe, added Britain's Lovell-Badge.

"What we do not want is for rogue clinicians to start offering treatments" that are unproven like has happened with some other experimental technologies, he stressed.

Among key questions: Would the technique work if mom, not dad, harbored the mutation? Is repair even possible if both parents pass on a bad gene?

Mitalipov is "pushing a frontier," but it's responsible basic research that's critical for understanding embryos and disease inheritance, noted University of Pittsburgh professor Kyle Orwig.

In fact, Mitalipov said the research should offer critics some reassurance: If embryos prefer self-repair, it would be extremely hard to add traits for "designer babies" rather than just eliminate disease.

"All we did is un-modify the already mutated gene."

Published at 1:33 PM EDT on Aug 2, 2017

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Scientists Repair Gene in Human Embryos for First Time - NBC New York