Turning Stem Cells in Testes into Testosterone-Producing Cells in a Sustainable Culture System


A research effort led by scientists at Johns Hopkins Bloomberg School of Public Health, in collaboration with researchers from Wenzhou Medical university of China has successfully made testosterone-producing stem cells in culture that can be propagated in the laboratory.

Haolin Chen of the Bloomberg School of Public Health noted that testosterone treatments often produce spikes and troughs in testosterone concentrations that can cause a variety of side effects. Administering testosterone-producing cells might very well prevent these wide variations in testosterone production and decrease the potential side effects. Low testosterone in males has been linked to increased mortality, in addition to depression, decreased cognition and immune function, increase body and reduced muscle mass, and poor healing.

A group of cells called Leydig cells in between the seminiferous tubules in the testes of males typically produce testosterone in response to stimulation by a hormone called luteinizing hormone (LH), which is made by the anterior pituitary. Leydig cells produce testosterone in a rather stable, constant fashion, in contradistinction to the injections that are given to males with low testosterone levels.

Unfortunately, keeping testosterone-producing Leydig cells or Leydig cell progenitors alive in culture has proven rather difficult. To address this problem, Chen and his collaborators started adding combinations of growth factors to the cells to determine if any cocktails of growth factors or nutrients could keep the cells alive. Fortunately, they came upon a combination of platelet-derived growth factor, basic fibroblast growth factor, activin, and a molecule called desert hedgehog that stimulated the proliferation of the Leydig cell precursors. Desert hedgehog and activin in general drove the differentiation of these cells into testosterone-producing Leydig cells.

Further work revealed a cell surface protein called CD90 that earmarked all the stem cells in the testes of rats that could be differentiated into Leydig cells.

Chen thinks that the primary culture-differentiation system that he and his colleagues have devised could serve as a useful model system for stem cells in general, or as a clinically relevant system that could produce testosterone-producing stem cells for males with low testosterone levels.

“Our work could eventually offer a whole new therapy for individuals with low testosterone,” said Chen.

This work was published in the Proceedings of the National Academy of Sciences USA, 2016; 113(10): 2666 DOI:10.1073/pnas.1519395113.

Bone Marrow Stem Cell Injections Restore Fertility In Mice Made Sterile by Chemotherapy


Every year, over 20,000 women of childbearing age are diagnosed with cancer. Cancer treatments often include chemotherapy regimens that damage other tissues and the ovaries and its eggs are particularly sensitive to such treatments. Consequently, many young, female, cancer survivors are infertile as a result of their cancer treatments, and suffer early menopause and ovarian failure.

Now an earth-shaking study by Egyptian and American scientists has shown that stem cell injections into the ovaries can rejuvenate them and restore the fertility of laboratory animals.

“This approach carries high promise to women with chemotherapy-induced and potentially other types of premature ovarian failure,” said Dr Sara Mohamed, lead researcher for this project.

Woman who must undergo chemotherapy are routinely advised to freeze their eggs before they undergo any cancer treatments. However this procedure is labor intensive and takes time, and in urgent cases, there is not enough time to preserve the patient’s eggs. This leaves the woman in the unsavory position of having to decide between her fertility or her life.

A procedure like the one used in this study might give female patients other options that do not force them to choose between the Scylla of their ability to have their own children and the Charybdis of their survival.

To date, this procedure has been successfully performed in laboratory mice. In this experiment, a clutch of eighteen laboratory mice were broke into three groups of six. One group of six female mice was treated with anticancer chemotherapeutic agents, followed by injections of bone marrow stem cells into their ovaries. The second group of six female mice also received chemotherapy, followed by injections of sterile saline into their ovaries. The third group, a control group, received injections of sterile saline into their ovaries without receiving prior treatments with chemotherapy.

One week after receiving their treatments, the stem cell-treated mice showed a significant increase in estrogen production. Since estrogen is a sex steroid hormone that is essential to ovulation, these results suggested that the menstrual cycles of the infertile mice was actually being reconstituted. Then a week later, mice in the stem cell-treated group showed regeneration of their ovarian tissue and increased numbers of ovarian follicles. Ovarian follicles produce the sex steroid hormones estrogen and progesterone and contain a single egg that matures during the follicular stage of the menstrual cycle and is potentially released during ovulation. These same mice, which had experienced ovarian failure as a result of chemotherapy, were able to mate with male mice, and eventually give birth to large litters of healthy mouse pups while those who had saline injections continued to suffer from reduced fertility of even infertility.

follicular-phase

These treatments worked so remarkably well, that the members of the researcher team who were involved with this project want to move to human trials as soon as possible.

Dr Sara Mohamed, of Mansoura Medical School in Egypt, who served as the lead researcher of this project, said she had come up with the idea after meeting a 22-year-old cancer patient who had a high risk of infertility from chemotherapy. Dr. Mohamed said: “It was a very emotional for me so I decided to pursue it and work on it to figure it out. It [is] a very common problem based on statistics of cancer female diagnosis every year. “

Dr. Mohamed continued: “We inject[ed] stem cells in[to] the ovaries of mice which had chemotherapy and were damaged and we got very good ovarian function restoration in form of follicle number, hormonal production, and finally getting pregnant and having new pups, which was our ultimate goal.  We are now working on translating that into clinical trials (for humans).  This approach carries high promise to women with chemotherapy-induced and potentially other types of premature ovarian failure.”

Imperial College gynecologist Stuart Lavery said: “This is very exciting piece of research that adds to our understanding of how cells differentiate to become egg stem cells.” Dr. Lavery served as a consultant on this research. I must add at this point as an aside that it is rather unlikely that the bone marrow stem cells are differentiating into eggs. Instead the bone marrow stem cells are probably augmenting the survival and health of existing eggs in the ovary.

Dr. Lavery continued: “Clearly, there remains an enormous amount of work to see whether these results would be transferable into humans. But it does provide some realistic hope that post-chemotherapy patients who have been made menopausal could one day restore ovarian function and possibly fertility.”

Dr. Mohamed and her colleagues would like to initiate human trials using umbilical cord or even embryonic stem cells. They will need to convince regulatory agencies that the procedures they have designed are safe. For this reason, I find it unlikely in the extreme that the US Food and Drug Administration (FDA) would give approval for an embryonic stem cell-based trial in the ovaries, given the large numbers of regulatory and safety hurdles other recent embryonic stem cell-based trials have had to conquer. Also, it is worth noting that the FDA has not approved other proposed trials that sought to stimulate ovarian-based stem cells. For this reason, getting FDA approval for their trial might prove difficult. Also, one mouse experiment is not going to be enough to persuade the FDA to acquiesce to their proposals. Large experiments will need to be done and large animals studies would also be needed as well.

Women who opt to freeze their eggs can use in vitro fertilization (IVF) to have their own children. Alternatively, if the eggs are fertilized with her mate’s sperm, then the embryos can development to the blastocyst stage after which they are cryopreserved (frozen) before chemotherapy for later family-building purposes.

Such a strategy leads to some problems in countries with nationalized medicine: some provinces have decreased funding for IVF, since IVF is very expensive and the demand is below the cost to maintain such faculties. Likewise, at times, female cancer patients are denied the option of cryopreservation, again because of the costs and the lack of a nearby facility that has the space, means, or funding to keep her embryos on ice for a time. A new regenerative therapy might give such a female patient some solace with regards to her future fertility.

A consultant in Reproductive Medicine and Surgery at Hammersmith Hospital, London, Dr Geoffrey Trew, said of this research: “Fertility-wise, if this works it would be stupendous. Certainly it does appear promising and anything you can do to regenerate and ovary is a good thing. Theoretically if you are regenerating the ovary you should be getting better quality eggs. Clearly we’re not here yet, and it’s good that the researchers are not over-claiming their findings, but it’s a great proof of concept.”

Dr Edgar Mocanu, consultant gynecologist at Rotunda Hospital in Dublin and a board member of the International Federation of Fertility Societies, said: “This could open phenomenal opportunities for women. Millions of women around the world undergo cancer treatment and some of them will become infertile through ovarian failure. While cancer survival rates have increased dramatically, to date there is no effective method of preventing infertility after chemotherapy. It could also open new avenues for the treatment of menopause induced health issues.”

Dr Owen Davis president of the American Society for Reproductive Medicine: “If this experimental treatment can be translated to women who have lost ovarian function from chemotherapy, it will be a great advance. Restoring ovarian hormone production, follicle development and fertility to chemotherapy patients is a potential new application for bone marrow donation that could help many women.”

Radio Interview About my New Book


I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

Adult Mammals Lack the Stem Cell Activity to Make New Eggs


Recent research in mice and humans have discovered a stem cell population in ovaries that can form eggs. However, this discovery begs a question: namely, why do adult female mammals run out of eggs in their lifetime if they have a stem cell population that can produce eggs?

New research from the Carnegie Institute for Science demonstrates that adult mice do not use stem to produce new eggs, thus answering this apparent conundrum.

Before birth, mouse and human ovaries contain an abundant supply of germ cells that originate from primordial germ cells that form from the inner layer of the primary umbilical vesicle (otherwise known as the yolk sac).  Between the time when the embryo is four to six weeks old, the primordial germ cells (PGCs) migrate from the wall of the primary umbilical vesicle to the gut tube.  From the gut tube, the PGCs migrate to the dorsal body wall by means of the mesentery that suspends the gut from the body wall.  Once in the body wall, the PGCs come to rest on either side of the midline in the loose mesenchymal tissue just inside the membranous lining of the body cavity (known more technically as the coelomic cavity).

PGC Migration Pathway2

Most of the PGCs populate the region of the body wall at the level that will form the gonads.  During their migration, PGCs continue to multiply by means of mitosis, which increase their numbers substantially.  Some PGCs may become stranded during their migration, coming to rest at extragonadal sites.  Occasionally, stray germ cells of this type may give rise to a type of tumor called a teratoma.

Teratoma
Teratoma

Once in their final location, the PGCs will stimulate the formation of the genital or gonadal ridge.

In females, PGCs (which are now called gonocytes) undergo a few more mitotic divisions after they are surrounded by the somatic support cells and become intimately associated with them.  The gonocytes differentiate into oogonia, and by the 5th month of fetal development all oogonia initiate meiosis.  After they initiate meiosis, the oogonia are called primary oocytes.  However, during an early phase of meiosis all sex cells enter a state of dormancy, and they remain in meiotic arrest as primary oocytes until sexual maturity.  Beginning at puberty, each month a few ovarian follicles resume development in response to the monthly surge of pituitary gonadotropic  hormones, but usually only one primary oocyte matures into a secondary oocyte and is ovulated. This oocyte enters a second phase of meiotic arrest and does not actually complete meiosis unless it is fertilized. These monthly cycles continue until the onset of menopause at approximately 50 years of age.

Near the time of birth, the ovaries of mice and humans contain an abundant supply of eggs that will be released from follicles during ovulation each menstrual cycle.  At the birth of the baby, she will possess a large reserve of primordial follicles that contain a single egg surrounded by supporting follicle cells.  Evidence of new follicle production is absent after birth.  Therefore, it has long been thought that the supply of follicles is fixed at birth and eventually is exhausted at menopause.

During the last decade, researchers have found primordial follicles in adult mouse ovaries that turn over and claimed that adult germ-line stem cells constantly resupply the follicle pool and sustain ovulation.  These claims were based on observations of ovarian tissue and one the behavior of extremely rare ovarian cells after these cells were cultured for some time in the laboratory.  Such criteria are subjective, especially in light of the fact that culturing cells for long periods of time in the laboratory can effectively reprogram them.

At Carnegie, Lei Lei and Allan Spradling used a technique that tracks individual cells and their progeny within living tissue over a specific time course.  The cells are marked with a gene, and this gene is inherited by the progeny of that cell, thus allowing the careful tracking of all the progeny of that cell or those cells.  This technique is called “lineage tracking” and it is a very popular technique in developmental and cell biology.

By subjecting primordial follicles to lineage tracking, Lei and Spradling showed that germ-line stem cell activity cannot be detected in mice.  Furthermore, primordial follicles are stable, and even if half the existing follicles die off, no germ-line stem cell activity is detectable.  This research does not prove that there are no germ-line stem cell divisions within the ovary of the mouse, but it does place an upper limit on the divisions of the germ-line stem cell population of one division every two weeks at the most, which is biologically insignificant.

What then can be said about the germ-line stem cell cultures isolated in the laboratory?  According to Alan Spradling, the cells “likely arise by dedifferentiation in culture,” and “the same safety and reliability concerns would apply as to any laboratory-generated cell type that lacks a normal counterpart” in the body.

This should be a warning to those conclusions that are solely derived from experiments conducted in culture alone and not in a living creature as well.

Stem Cell Transplants Restore Fertility in Monkeys


Injections of banked sperm-making stem cells can restore fertility to male non-human primates. This work comes from stem cell researchers at the University of Pittsburgh School of Medicine and the Magee-Womens Research Institute and was published in the journal Cell Stem Cell.

This is a remarkable finding because some men or boys must undergo cancer treatment before they have their families. Since cancer drugs destroy dividing cells and do not discriminate between normal cells and cancer cells, the stem cells that make sperm tend to take a serious beating during chemotherapy. The cancer might be destroyed, but the patient will be rendered sterile.

The senior investigator for this work, Kyle Orwig, associate professor in the Department of Obstetrics, Gynecology and Reproductive Medicine at the University of Pittsburgh School of Medicine said: “Men can bank sperm before they have cancer treatment if they hope to have biological children later in their lives,” he says. “But that is not an option for young boys who haven’t gone through puberty, can’t provide a sperm sample, and are many years away from thinking about having babies.”

Young boys that have yet to experience puberty do not yet make any sperm, but they have a modicum of spermatogonial stem cells in their testes that are waiting in the wings to produce sperm during puberty. During puberty, neurons in a part of the brain called the hypothalamus release a 10-amino acid peptide called “gonadotropin releasing hormone” (GnRH). Because these neurons release their GnRH into blood vessels that feed the pituitary gland, just below the hypothalamus, it flows directly to the pituitary.

GnRH stimulates the anterior lobe of the pituitary to release two trophic hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are collectively known as gonadotropins. FSH initiates sperm cell production in the tubules within the testes and LH stimulates the synthesis of the steroid hormone testosterone, which is necessary for sperm maturation.

Orwig and his colleagues wanted to determine if it is possible to restore fertility by freezing and banking these spermatogonial stem cells and then reintroducing them back into the testes after the completion of chemotherapy. Orwig and others took biopsies from the testes of young, adult male macaque monkeys that had yet to experience puberty. This tissue was cryopreserved in small samples. Then the monkeys were treated with chemotherapy agents that are known to reduce fertility.

A few months after chemotherapy treatment, Orwig and his colleagues transplanted each monkey’s own spermatogonial stem cells into their own testes by means of ultrasound-guided surgery. Nine of twelve adult animals showed restoration of sperm production and three of five very young animals that had not yet experienced puberty demonstrated an ability to make functional sperm after they reached maturity.

In a second experiment, spermatogonial stem cells from unrelated monkeys were transplanted into infertile animals. These transplanted cells generated sperm that had the DNA fingerprint of the donor. Because the testes contain a barrier to the immune system that prevents access of the sperm to the immune system, the implanted tissue could survive without being attacked by the immune system. This is a problem is males who have immune responses to sperm. For example, men who have had a vasectomy or make homosexuals have immune responses to human sperm. Laboratory tests showed that sperm from transplant recipients successfully fertilized 81 eggs that lead to embryos that developed normally. Donor parentage was confirmed in these embryos.

“This study demonstrates that spermatogonial stem cells from higher primates can be frozen and thawed without losing their activity, and that they can be transplanted to produce functional sperm that are able to fertilize eggs and give rise to early embryos,” Orwig says.

Several centers in the U.S. and elsewhere are already banking testicular tissue for young male cancer patients. This is in future anticipation that new stem cell-based therapies will be developed that will help them achieve pregnancy and have their own biological children. Thus this proof-of-principle experiment has generated no small degree of excitement for clinicians and patients who have compromised fertility.

According to Orwig, “These patients and their families are the pioneers that inspire our research and help drive the development of new medical breakthroughs.” He continued: “Many questions remain to be answered,” Orwig notes. “Should we re-introduce the spermatogonial cells as soon as treatment is over, or wait until the patient is considered cured of his disease, or when he is ready to start a family? How do we eliminate the risk of cancer recurrence if we give back untreated cells that might include cancer cells? These are issues we still must work through, but this study does show us the concept is feasible.”