The Cellular Time Machine
Published in Lab Times 06-14.
Somatic cell reprogramming – the tuning back of highly specialized cells to their naïve embryonic “stem cell” state – is the future of medicine. However, advances in the field are precluded by gaps in our knowledge of stem cell biology. Maria Cosma’s discovery of a novel ‘molecular wave’ in stem cells fills up a major void.
On February 22, 1997, newspapers and TV channels world-wide scurried to announce a major scientific breakthrough: the cloning of the world’s first mammal, Dolly the sheep. Unlike most animals which develop from a sperm-fertilized egg, Dolly was born without a dad – simply out of an unfertilized egg, the nucleus of which was swapped with the DNA from a mammary gland cell of a Finnish Dorset ewe. Dolly’s development bypassed genetic exchange processes between maternal and paternal DNAs. Instead, what she had was the intact genome of the ewe, making her the first ever mammalian clone.
All vertebrates start out as a mush of embryonic stem cells (ESCs) in the womb. ESCs are pluripotent, in other words plastic – they can proliferate and morph into any ‘somatic’ cell of the adult animal such as a nerve cell, a liver cell, and so on. But over the course of development, the cells become less versatile, they stop dividing and differentiate into a specific cell type, after which there is little turning back – for instance, a nerve cell does not spontaneously switch types to become a liver cell, or vice versa. At the outset, the process appears to be irreversible – a fully differentiated cell loses its plasticity ostensibly by turning off all other genes except the ones that define its type. But the success of Dolly and many similar experiments have proved quite the opposite. The DNA of a specialized cell viz. mammary gland cell still retains a memory of its past, and can turn back time when inserted into an egg to give rise to naïve stem cells and consequently, an entire organism.
Triggering the cellular time machine, a technology dubbed somatic cell reprogramming (SCR), relies on exposing mature cells to an altered milieu resembling that of a nascent embryo. SCR is attractive to medicine given its potential clinical applications – de-differentiated patient cells, differentiated artificially into any cell type, can offer insights to diseases, or generate whole tissues for safe grafting in cases of organ failures. While a gust of discoveries have shed light on the factors that spark SCR, such as transcriptional regulators, what yet remains a mystery is the nature of ensuing molecular events that reinstate a specialized cell to an ancestral stem cell. Maria Pia Cosma, the principal investigator of the Reprogramming and Regeneration group at the Center for Genomic Regulation (CRG) in Barcelona, Spain, has over a decade’s worth of findings on the molecular mechanisms of SCR and ESC pluripotency. Her research on Wnt signaling in reprogramming was recently published (Cell Reports vol.8:1-11).
Turning back time
Maria Cosma started her career in the lab of Andrea Ballabio at the University of Naples, Italy, where she probed into the genetic basis of multiple sulphatase deficiency (MSD) disorders, a spectrum of diseases in humans caused by the inactivation of enzymes called sulphatases. “MSD is a multisystemic disease in which the substrates that are normally broken down by sulphatases build up in different tissues”, Maria describes. In patient cells grown in a dish, the substrates accumulate causing massive cell death. Using a technique called microcell-mediated chromosome transfer, Maria could rescue the function of the enzyme in the MSD cells. “We transferred the chromosomal content of a healthy cell line only to find that it jolted up sulphatase activity in the patient line”, she recollects.
Following her initial success with genetic complementation in patient lines, Maria’s interests soon crystallized on SCR, an emerging technology with profound therapeutic potential. “I started working on cell-fusion based reprogramming as a side project during my postdoc in Naples, and then continued with it in my own lab at the CRG”, she remarks. Similar to the nuclear transfer method used in cloning Dolly, cell-fusion resets the clock in differentiated cells to stem cell stage but here “it comes from fusing two intact cells, a stem cell and a somatic cell. The results of such fusions are pluripotent cell hybrids”.
When somatic cells fuse with stem cells, they bump into transcriptional regulators in the latter that jumpstart previously silenced ‘pluripotency’ genes in the differentiated cells. One such transcriptional regulator is Nanog, a protein abundant in ESCs. Nanog is thought to be a central organizer that imposes pluripotency in somatic cells in cell-fusion experiments (Nature vol.441:997-1001).
Wnt pathway resets the clock
Using the cell-fusion approach, Maria and colleagues successfully instated pluripotency in a number of specialized cells. “In one instance, we injected bone marrow stem cells into a damaged mouse retina. The retinal neurons at first reprogrammed to an embryonic state as they swiftly fused with the stem cells. These hybrids proliferated and then differentiated into mature neurons that repopulated the retina and partially mitigated the damage”, the geneticist recollects. Importantly, in their experiment, the stem cells had to be pre-treated with an activator of the Wnt pathway, a crucial molecular signal that endows ESCs with the capacity of self-renewal and pluripotency (Cell Reports vol.4(2):271-86).
Being the curious person that she is, Maria was immediately intrigued by her findings – the indispensability of Wnt in reprogramming, “I’ve known that Wnt signaling oscillates during mouse embryonic development, but I asked how Wnt functions in cell-fusion based reprogramming and how it maintains ESC pluripotency”.
In a battery of experiments, the group first confirmed that Wnt activity is critical for pluripotency. They probed for β-catenin, a readout of Wnt activity, in Nanog-rich mouse ESCs – a gold standard for pluripotency – cultured in a dish. These cells indeed exhibited high levels of β-catenin, suggestive of heightened Wnt activity. β-catenin expression strongly correlated with the levels of Nanog itself and was maintained by Nanog-mediated suppression of a negative regulator of Wnt pathway. Next they asked whether Wnt activity is pivotal to cell-fusion based reprogramming. In cell fusions of neural cells with ESCs, Nanog fostered reprogramming of the hybrids only when Wnt activity was intact. Ablating Wnt signaling by introducing negative regulators or by deleting β-catenin interfered with SCR. Moreover, cell fusions of Nanog-deficient ESCs and neural cells still showed successful reprogramming if β-catenin levels were upped by Chiron, a molecule that blocks β-catenin breakdown. In a nutshell, Wnt activity alone is critical for SCR and Nanog positively reinforces Wnt signaling to promote pluripotency of ESC hybrids.
The molecular wave
How does Wnt activity facilitate and maintain ESC pluripotency? To answer this question, the Spanish group turned to analyze the dynamics of Nanog and β-catenin levels in ESCs grown in two types of culture media, (i) serum+LIF, normal media that promotes ESC proliferation and differentiation, and (ii) 2i+LIF, media rich in Chiron that keeps cells in continued pluripotency. “In cells cultured in serum+LIF, we found that β-catenin levels oscillate and is closely linked with Nanog, which also fluctuates similarly. But in 2i+LIF, while Nanog ceases to oscillate, β-catenin continues to do so. This, we think, may be important to maintain pluripotency”, Maria describes her findings. In normal media, Nanog levels oscillate owing to random gene expression changes and β-catenin expression closely follows suit. But in 2i+LIF, β-catenin fluctuates independently of Nanog, whose levels are rather homogeneous. Such discrepancy in their dynamics may explain why serum+LIF allows differentiation – perhaps, during the trough of Nanog expression, while 2i+LIF with steady Nanog levels protracts pluripotency. “In essence, oscillations of β-catenin may be the answer to maintaining pluripotency. They present stem cells with multiple choices – the propensity to differentiate into any cell type. It’s unlike having just two choices, as when a gene is ON or OFF”, explains Maria.
Back to future
The Cosma lab’s work uncovers a novel mechanism that steers ESC pluripotency, but future work may have to address whether and how the waves of Nanog or β-catenin expression enable reprogramming of somatic cells. A good place to look for answers would be target gene expression profiles. Maria not only intends to delve into the mechanics of stem cell culture but also hopes to take a step back and try out current methodologies in tissue regeneration. “We use stem cells to alleviate brain degeneration in a mouse model of Parkinson’s. Moreover, we are testing the effects of hybrid cells in liver regeneration”, she enlists ongoing projects. “We are also advancing in the structural front. In a collaboration with physicists, we use high-resolution imaging techniques to visualize chromatin structures, and get an estimate of the pluripotency grade of stem cells”, she concludes.