Research

Given the longstanding interest and expertise of Dr. Sebastiano in embryonic development, germ cells biology, and stem cells, we think we are uniquely positioned to look at these complex and challenging goals by taking a holistic and comprehensive approach that is focused on learning how normal embryonic and fetal development occurs with the goal to hijack and re-purpose these principles for the cure of aging, aging associated diseases, and degenerative and developmental disorders.

It is difficult to draw a demarcation line between the germ cells, the embryo, the fetus, and the adult. Development happens as a continuum. Good germ cells generate good embryos, fetuses and healthy individuals who will generate germ cells, that will generate new individuals, over and over again. This is the reason why we study embryonic development and germ cells. Because they are the key to understand how information is passed to the next generation, how it is reset to zero in terms of developmental potency and aging, and how it gets re-organized to form organs and tissues.

The beginning of any individual’s life starts with fertilization, the fusion of a male and a female gamete, known as sperm and egg (or oocyte).  This event delineates a very important phase of transition in cell identity and cell function that turns two fully differentiated cell types (the gametes) into a brand-new cell type called the zygote from which all of the trillion cells we are made of originated from. How is this possible?

Soon after fertilization a massive reprogramming of the maternal and paternal chromatin occurs. This affects the DNA methylation, histone positioning, histone modifications and much more; the germ cells chromatin gets “reprogrammed” or “reset” into a new chromatin that is then ready to initiate a new developmental program that will lead to the formation of a blastocyst, of a gastrula, and then further on to adult life. Understanding this process of reprogramming is vital. Why? Because by understanding it we can learn how to implement it to turn one cell type into another cell type and turning and aged cell into a young cell.

In fact, a zygote is not only a new cell type, it is also the youngest cell type. If any age (in the form of epigenetic drift) that is accumulated in the germ cells was not reset, then this would progressively accumulate and lead to a species crash that would result in death. Instead, every time a new zygote forms, the epigenetic inheritance of the germ cells is reset to i) establish a new program of development, and ii) to establish an age zero offspring.

In the lab we are studying this phenomenon using a multitude of approaches. We study human and mouse preimplantation embryos and use human pluripotent cells to model this phenomenon. For example we have recently identified a class of novel long non coding RNAs that have retroviral origin and shown that they are important to lead to the formation of pluripotent cells both in vivo (in the human embryo) and in vitro, during the formation of iPSCs.

Formation of ovary and testis during human embryonic development time point has implications for understanding infertility and endocrine diseases. Since, it is not possible to study gonadal development in humans at embryonic stage in vivo, we can study their development through differentiation of human pluripotent stem cells in vitro. Our preliminary data indicates that germ cells can be specified and differentiated from human ES cells. We would also like to achieve differentiation of female gonadal supportive cell types, known as granulosa which are important for ovarian development and function in adults.

Over the past century, greater medical knowledge and technical advances have improved the quality and duration of human lives. Humans are unique in that they experience exceptionally long post reproductive lifespans, and the reproductive status influences the health of an individual throughout the chronological life span, especially in women.

Recent studies lend credence to old theory that longevity is tied to fertility. DNA analysis of 270,000 volunteers show hundreds of gene mutations boosting fertility are also correlated with shorter life span. Gene variants impacting reproduction are almost five times more likely to influence life span. However, very little is known about how these genes and their variant are transcriptional and epigenetically regulated at cellular and molecular levels. The scope of the project in the lab is to characterize how the ovarian input systemically affects the transcriptional and epigenetic changes in multiple tissues of young and aged female mice and modulate those epigenetic changes by reprogramming as an intervention strategy to maintain and improving the quality of oocytes and functionality of ovaries that would preserve and improving the quality of life and lower the risk of other diseases.

Almost 20 years ago, the regenerative medicine field reached a milestone when Dr. Yamanaka's discovered that only 4 transcription factors, named Oct4, Sox2, Klf4 and cMyc, were enough to convert an adult specialized cell in an induced pluripotent stem cell (iPSC). This redirecting of a fully specialized cell into an induced pluripotent stem cell not only reacquires the pluripotent capability but also removes the hallmarks of aging to zero. Since then, many researchers have been working on different strategies for rejuvenation by partial reprogramming that permits reverting the aged cell phenotype without losing its identity. A few years ago, at our lab, we proved that a temporary expression of the Yamanaka factors in human cells from old donors diminishes cellular aging.

Now, we aim to understand the molecular mechanisms of partial reprogramming involved in the rejuvenation phenomenon among different cell types. To achieve this goal and for each cell type, we are studying how the Yamanaka factors bind to the chromatin and what process they trigger in the DNA methylation, RNA expression, and chromatin structure. These studies will help us to discern, if possible, the differentiation process from the rejuvenation one, in order to find new targets and develop novel therapies for age reversal.

As we age, our susceptibility to diseases and tissue dysfunction increases, impacting our overall well-being. To address this, we are exploring two promising strategies: Reprogramming-Induced Rejuvenation (RIR) and senescent cell removal through senolytics.

RIR reverts natural aging by induction of the four Yamanaka factors, transcription factors used for generating induced pluripotent stem cells. One disadvantage of this strategy is its high risk of teratoma formation. Senolytics, on the other hand, aim to eliminate aged cells but can have side effects. A recent study in drosophila suggested that combining RIR with senolytics could enhance rejuvenation, but this hasn't been explored in human or mouse systems.

Our research seeks to understand the molecular intricacies of combining RIR and senolytics to create a safe anti-aging therapy. We hypothesize that senescent cells contribute to the loss of cell identity during reprogramming, and removing them may enhance safety. Our experiments involve in vitro studies, wound healing models, and in vivo mouse experiments to test these hypotheses.

By deciphering the role of senescent cells and optimizing the combination of RIR and senolytics, we aim to create a cancer-safe anti-aging therapy. This research has profound implications for reducing age-related diseases and improving overall health, contributing to a brighter and healthier future for all.

TBX1 is closely linked to the 22q11.2 Deletion Syndrome (22q11.2DS), the most common of the microdeletion syndromes, which is caused by hemizygous loss of 0.7-3 Mb of DNA on chromosome 22 and results in a constellation of clinical phenotypes. The core phenotype originates from disrupted development of the pharyngeal apparatus. Although approximately 50 genes may be deleted, it is the haploinsufficiency of the transcription factor TBX1 that recapitulates most of the critical phenotype associated with 22q11.2DS. The overarching hypothesis is that TBX1 is at the center of a Gene Regulatory Network critical for both the formation and maturation of the pharyngeal endoderm and the morpho-patterning of the surrounding mesoderm and neural crest cells.

In the lab we have recently developed and characterized an in vitro model which faithfully mimics the formation and progression of human pharyngeal endoderm, thereby providing an unprecedented opportunity to tease out the functions of TBX1 in its physiological context. Specifically, this model will be used to identify the transcriptional targets and partners of TBX1, to investigate the role of TBX1 as an epigenetic regulator of the human pharyngeal endoderm, and to mechanistically investigate newly discovered putative regulatory regions of the TBX1 locus.

The proposed work is expected to identify the molecular mechanism at the basis of TBX1 haploinsufficiency and identify pathways that could be rescued through pharmacological intervention. Dissection of the epigenetic and molecular machinery responsible for pharyngeal endoderm formation will be instrumental in informing the generation of cell therapies for 22q11.2DS.

Epigenetic partial reprogramming with OSKM has demonstrated impressive rejuvenating effects in multiple types of human primary cultured cells. One of our current research goals is to build a translational bioresearch bridge to apply partial reprogramming as a tool to treat age-related diseases or regenerative challenges. To do this, we are using a complex ex vivo organoid as well as transgenic animals in vivo models to study skin aging-related pathophysiological conditions and diseases, especially delayed skin wound healing.