Balancing act

Dr Robin Hobbs* got off to a stellar start with a breakthrough finding published in Nature. Now he’s continuing to investigate a delicate balancing act that occurs within stem cells in the testis.

By the very nature of scientific research, which often mimics the convoluted and complex pathways it studies, many scientists end up following a circular research route, often unintentionally and usually over the span of many years. Dr Robin Hobbs, a relatively new addition to the Australian Regenerative Medicine Institute (ARMI) at Monash University, managed to do it within a year or so of finishing his PhD, and rather spectacularly at that, with a landmark finding published in Nature Genetics.

After undergraduate studies at the University of Oxford, Hobbs completed his PhD at University College London in 2003 under the guidance of stem cell guru Professor Fiona Watt. He then moved fields and countries to take up a postdoc first at Memorial Sloan-Kettering Cancer Center, then at Harvard Medical School, with Professor Pier Paolo Pandolfi, a leader in the field of cancer genetics.

There he planned to work on the molecular mechanisms underlying leukaemia. However, he was immediately thrust straight back into the world of stem cells with his first major experimental finding. Characteristic of many scientific breakthroughs, Hobbs serendipitously managed to identify one of the first transcription factors needed to regulate germ-line or spermatogonial stem cells in the mouse testis.

When Hobbs joined the Memorial Sloan-Kettering Cancer Center lab in New York as a postdoc in 2003, the team there was working on the transcription factor promyelocytic leukaemia zinc finger (PLZF) because of its reported association with chromosomal abnormalities in rare cases of leukaemia.

“We wanted to model the human disease, so we made a PLZF-knockout mouse in the hope of seeing leukaemia or some sort of solid tumour develop,” says Hobbs. “However, one of the most striking phenotypes of this mouse was actually male infertility. We saw a loss of germline stem cell self-renewal capability in the knockout mice followed by a progressive depletion of germ cells and, eventually, infertility.”

Hobbs subsequently found that while expressed at moderate levels in haemopoietic cells, PLZF is expressed strongly in the spermatogonial stem cells in the testis in both mouse and human, making these stem cells his new obsession.

Maintenance of a wide array of adult tissues is dependent on a resident population of stem cells that must self-renew and generate differentiating daughter cells. However, at that time, very little was known about how the mitotic spermatogonia of the testis manage to continually self-renew and differentiate into sperm over much of the adult lifetime. Even less was known about the testis-resident stem cells that generate differentiating spermatogonia, the spermatogonial stem or progenitor cells (SPCs).

“This pool of cells within the testis is required for lifelong maintenance of the male germ line cells and, of course, for maintaining fertility,” he says. “SPCs have all the typical characteristics of adult stem cells: they reside in the tissue; are able to self-renew; have a high proliferative potential; and are able to generate differentiating spermatogonia, which then go on through meiosis to generate sperm. These stem cells are also heavily dependent on their unique niche within the testis.”

Cultured cells

Based on this new and exciting finding about the life and times of SPCs, and building on the few other reports of factors needed for SPC growth, Hobbs spent the rest of his postdoc studies developing a mouse testis model system for isolating, growing and maintaining his new favourite cells in culture so they could be studied further.

Due to the work of a number of pioneers in the germ cell field, including Hobbs, SPCs could now be grown and monitored for relatively long periods of time - up to one year - under highly specialised cell culture conditions while still maintaining their full developmental potential. At the time, this technical advance made the testis stem cell model unique among the commonly studied adult stem cell systems such as bone marrow, neural or skin. “We ended up with a very useful system for studying stem cells,” says Hobbs.

Primarily, the cell culture model allows scientists to interrogate SPC behaviour and regulation by in vitro techniques such as biochemistry that require large numbers of cells. “Then, at any time, we can assess the stem cell potential in vivo by transplanting the cultured cells back into recipient mouse testis, which have been depleted of endogenous germ cells, and looking for differentiation into sperm. So you have a very nice in-built assessment of your cells in culture.”

Hobbs continued to develop these experimental systems to purify and culture the SPCs through his postdoc years as a critical part of finding out more them. He also hoped to identify novel functions of PLZF and other gene regulators that could then potentially be translated into other adult stem cell systems important to human disease. According to Hobbs, while the PLZF finding was undoubtedly important, it really was just a very simple observation of an unexpected role in male germ cell differentiation, and just the start of finding out what makes the SPC system tick.

One of the central questions for Hobbs to address was how the SPCs integrate all the signals in the testis. Some tell them to keep self-renewing and maintaining the stem cell pool while others tell them to get on with the job of differentiating into sperm. Indeed, this is a question of importance to all adult stem cell systems. “The appropriate control of stem cell self-renewal and differentiation is critical for tissue homeostasis while disruption of the balance between these processes can contribute to tissue degeneration or cancer,” he says.

“Using our testis model and the PLZF knockout mice, we then went on to identify a couple of key downstream targets of PLZF in the SPCs that are important in regulating their function,” says Hobbs. “One of these turned out to be a signalling pathway pivotal to cell growth, the mTORC1 complex.” mTORC1, or the mammalian target of rapamycin complex 1m, was the topic of his talk at the Hunter Meeting.

mTORC1 is a large and ubiquitously expressed signalling complex that promotes protein translation and, through this, controls multiple cellular functions including organelle synthesis, metabolic and biosynthetic pathways, and energy use by the cell. “Basically, mTORC1 controls most of the key pathways needed for cell growth. If you activate mTORC1, the cells grow. Upstream, mTORC1 integrates a whole variety of signals, so it is turned on in response to many different stimuli that a cell faces including nutrient availability, energy status, growth factors and cellular stress.”

In other words, the mTORC1 pathway, and therefore cell growth, is turned off in conditions that are not favourable, such as stress, but turned on in the good times, when nutrients are plentiful and the right growth factors are present. Thus, mTORC1 controls the pivotal cell question of whether or not to grow.

Keeping the balance

This complex is well studied and is dysregulated in many human illnesses, particularly cancer and metabolic disease, which is not surprising given its pivotal role in the cell. “Importantly for my work, mTORC1 is also very important for stem cell function, which has been increasingly recognised in the last few years,” says Hobbs.

If you aberrantly activate mTORC1, inevitably this is detrimental to maintenance of the stem cell pool. It is easy to imagine that anything that strongly promotes cell growth is bad for keeping stem cells in their place, so to speak, and eventually you get depletion of the population. “So, it is critical to correctly regulate mTORC1 in the SPC system.”

What Hobbs found in his knockout mice lacking PLZF expression was abnormally high mTORC1 activity in the SPCs. In trying to find out why this was happening, he showed that PLZF could transcriptionally regulate some of the upstream regulators of the mTORC1 pathway. In other words, PLZF indirectly regulates mTORC1 signalling to control cell proliferation. The next questions Hobbs and his colleagues addressed were why is it bad to have aberrant activation of mTORC1 in the SPCs, and whether they could develop models to correlate the perturbed signalling activity with the effects on stem cell maintenance and fertility.

A key observation in answering these questions was that high levels of mTORC1 activity activates negative feedback effects and inhibits the cell’s response to a crucial growth factor, glial cell-derived neurotrophic factor (GDNF). This factor is secreted by other cells within the testis niche and is absolutely required by the SPCs for self-renewal and growth. Indeed, SPCs cannot grow in culture without GDNF.

“So, we found that PLZF promotes SPC self-renewal through its ability to inhibit mTORC1, and in doing, increase the sensitivity of the cells to GDNF. In this context it could be an important mechanism to maintain stem cell homeostasis, ultimately to balance growth against self-renewal and maintain the pool of stem cells within the testis.”

Now based at ARMI in Melbourne, and with the mTORC1 findings published in Cell in 2010, Hobbs is continuing his search for other downstream targets of the mTORC1-PLZF axis in SPCs. He is interested in finding out how mTORC1 can control signalling through the GDNF receptor pathway and the effects of this on stem cell function.

“I also want to further explore the crosstalk between the different targets of mTORC1 and identify those co-regulated by PLZF and other genes to control cell fate. At some level everything talks to each other and it is just a question of working out what is required and what is not.”

An important conclusion in terms of translating these findings, according to Hobbs, is that changes in mTORC1 activity can change cell fate. “For example, if you have high activity the cells can differentiate as opposed to self-renew. So inhibitors to the TORC1 pathway might be one way to disrupt that balance and influence stem cell function to, for example, improve tissue regeneration.

“Of course, the details are somewhat complex because a certain amount of mTORC1 activity is needed for normal cell growth, so you would need something like a treatment regimen that would partially or temporarily inhibit the signalling in a way that would change the fate decisions of these stem cells and leave them to self-renew more, but then of course also allow them to grow and differentiate correctly.

“I would also really like to start looking at whether these findings in the SPC system could be translated to other adult stem cell compartments, such as the haematopoietic system, back where I started.”

*Dr Robin M Hobbs is a pioneering young researcher in the germ cell field. He recently relocated from Beth Israel Deaconess Medical Center associated with Harvard Medical School in Boston and has a joint appointment with Monash Immunology and Stem Cell Laboratories and the Australian Regenerative Medicine Institute (ARMI). His research is supported by NHMRC funding and by a Monash University Larkins Fellowship.

Image credit ©iStockphoto.com/Alexandr Mitiuc