Severe burns, trauma, and chronic wounds can result in extensive skin loss, leaving patients with few effective treatment options. Skin grafts from donors are often limited in availability and may be rejected by the patient’s immune system, while engineered skin substitutes rarely restore key functions like sweat glands or hair follicles. To address this need, we aim to create universal, off-the-shelf skin grafts that can be used in any patient without the need for immunosuppression.

Our approach uses human induced pluripotent stem cells (iPSCs) that are genetically engineered to be “hypoimmune”—meaning they are less likely to be attacked by the recipient’s immune system after transplantation. These hypoimmune iPSCs will be differentiated into early skin-forming cells and tested in a rat embryo model that lacks its own skin. This in utero model offers a unique environment to evaluate human skin development before transplantation.

We have previously demonstrated that human skin-like structures can be generated by injecting human keratinocyte cell lines into skin-deficient mouse embryos. In this new study, we aim to extend these findings by using stem cell–derived cells and moving from mice to rats, which provide a longer developmental window. This is an essential step toward testing in larger animals, such as pigs, which more closely resemble human skin in size and structure.

By combining immune engineering with developmental biology, our project lays the groundwork for a future where lab-grown, immune-compatible skin grafts could be available to all patients—offering safer, more effective healing for those with severe skin injuries or genetic disorders.

Methods to perform gene expression profiling of single cells have revolutionized stem cell and cancer research. Such methods are able to profile thousands of cells at time, with expression of thousands of genes detected in each cell. Cancer often arises due to mutations (changes in the DNA) in stem cells that give that mutated cell a fitness advantage over other cells. One limitation of current single-cell profiling methods is the lack of information obtained about DNA mutations. This is necessary to allow investigators to distinguish the mutant stem cells from the normal ones to see how the mutant cells differ. Current methods to identify the mutant cells are laborious and technically challenging and usually involve destroying the cells to release their molecules into a solution. To overcome these limitations, we have developed highly innovative methods that perform the key steps of the assays directly within the cells, something which has never been attempted before. This allows us to select the cells that have the right region of DNA amplified prior to the downstream steps, which greatly increases the efficiency of finding the mutant cells. In this proposal, we will further develop this technology and apply it to stem cells from people with blood cancers or those at high risk of developing blood cancers in the future. This will allow us to examine the gene expression programs in the mutant stem cells, which may reveal how the mutant stem cells gain advantages over the normal ones. Once the technology is further optimized, we anticipate that it will be broadly applicable to any cell types or cancers for which mutational profiling may be useful.

The spinal cord is crucial for breathing, movement, and other life-sustaining processes. In particular, there are special neurons within the spinal cord—known as phrenic motor neurons—that are crucial for breathing. This tiny group of neurons is necessary for life. However, these phrenic motor neurons are destroyed in a deadly disease known as amyotrophic lateral sclerosis (ALS, otherwise known as Lou Gehrig’s disease). Consequently, ALS patients gradually lose the ability to breathe, and therefore die. However, it is impossible to isolate phrenic motor neurons from patients, which has limited our ability to study ALS and to develop new therapies.

We are trying to create different types of human spinal cord neurons—including phrenic motor neurons—in a Petri dish. The ability to create phrenic motor neurons in a Petri dish will allow us to discover in greater detail how ALS affects these life-sustaining neurons, which could pave the way for new therapies to treat ALS and perhaps other neurodegenerative diseases that kill spinal cord neurons. Our efforts to create spinal cord neurons, including phrenic motor neurons, are fueled by our new ideas about how the spinal cord arises in development, and more specifically, that the spinal cord itself may emerge from two different types of progenitor cells.

Taken in collective, this project will enrich our knowledge of how the spinal cord arises during embryonic development, and we will use these insights to create human spinal cord neurons—including phrenic motor neurons—in a Petri dish.

Understanding the mechanisms driving age-associated neurodegeneration and developing strategies to reverse or mitigate it holds significant therapeutic potential for improving age-related neurodegenerative diseases. However, investigating brain aging and rejuvenation directly in mammalian systems presents considerable challenges.

This research proposes the colonial tunicate, Botryllus schlosseri, as a robust model system. As a close invertebrate relative of vertebrates, Botryllus shares significant genetic homology with humans, including genes crucial for brain development and implicated in neurodegenerative conditions. Its unique biology, featuring a complete weekly regeneration of its central nervous system via an asexual budding cycle and the persistence of long-lived stem cells, offers an unparalleled platform to study recurrent neurogenesis and stem cell aging. Notably, aging Botryllus colonies exhibit phenotypes that parallel mammalian aging, including reduced regenerative capacity and altered expression of neurodegeneration-associated genes like APP in their brain.

Our preliminary data confirm the presence of distinct brain cell populations in Botryllus, including candidate Neural stem cells (NSCs). Crucially, we have demonstrated that Pulsed Electrical Current (PEC) can reverse organismal aging phenotypes in Botryllus, enhancing stem cell-mediated regeneration and extending lifespan.

This study hypothesizes that PEC can directly rejuvenate aged Botryllus NSCs by modulating their intrinsic signaling and transcriptional activity, thereby restoring a more youthful and functionally robust CNS. We will apply PEC to aged colonies and employ cellular (flow cytometry, confocal microscopy) and molecular (spatial transcriptomics) analyses to assess rejuvenation at the NSC level. Reciprocal transplantation assays of candidate NSCs between PEC-treated and untreated organisms will further dissect cell-intrinsic versus niche-mediated effects of the electrical stimulation.

Most of what we know about mechanisms of early mammalian development comes from the studies of the mouse embryo. However, humans significantly differ from mice in many aspects of early development, which underscores the importance of developing genetically accessible, scalable and ethical models in which such human-specific features of development can be systematically examined. This is important, given that an estimated 60% of human conceptions fail around implantation into the uterus, with important implications for reproductive health and assisted reproductive technologies. Recently developed stem cell-based human 3D embryo models offer a unique opportunity for functional studies of the primate- and human-specific features of development. One such model, called ‘blastoids’ recapitulates morphology and formation of the three lineages of the human blastocyst, a human embryo stage just prior to implantation. However, to date systematic genetic or epigenetic perturbations have not been established in the blastoid system, thus far limiting its use to observational studies or small molecule-mediated interventions.

In preliminary studies, we established targeted genetic and epigenetic manipulations in the blastoid model to study human- and primate-specific aspects of early development. With these manipulations, we discovered a human-specific regulatory mechanism mediated by a novel, primate-specific transcription factor, whose expression during preimplantation is regulated by a retrotransposon insertion that is unique to humans. We further showed that this human-specific mechanism is essential for regulation of fundamental cellular processes, and for endowing human naïve pluripotent stem cells with blastoid-forming potential. In studies proposed here we build on these findings and leverage functional genomic and single cell approaches in stem cell-based 3D embryo models to study molecular mechanisms by which the discovered evolutionary young transcription factor gained developmental essentiality in humans. We will further conduct a genetic screen to systematically uncover other primate-specific genes that are essential for blastoid formation and lineage specification. The proposed studies will pave the way for systematic discovery of human- and primate-specific mechanisms that govern early development.

This research aims to understand a critical, yet mysterious, part of early pregnancy: how a human embryo attaches to the mother's lining of the uterus, the endometrium. This process, known as implantation, happens in the first two weeks after fertilization and is essential for a successful pregnancy. However, for many, implantation fails, which is a major reason why in vitro fertilization (IVF) can be unsuccessful. Up to 60% of high-quality IVF embryos may not implant, leading to early miscarriages and significant emotional burden for families, requiring multiple expensive IVF cycles. Problems at this early stage might also lead to later pregnancy complications such as preeclampsia. Scientists call this early stage a “black box” because it occurs hidden inside the uterus, making it difficult to study directly. Current knowledge is very limited and based on rare human samples, while animal studies may not be relevant because implantation differs greatly between humans and other mammals. To explore this “black box”, the Molè lab has developed an advanced model that closely mimics the lining of the human uterus, allowing to simulate the process of human embryo implantation outside the body. Working in collaboration with the Qiu lab, the goal of this project is to create the first-ever spatial atlas of gene expression of human embryo implantation. Using advanced transcriptomic technologies, this study aims to create a detailed 3D map of which genes are active in specific cells at the exact point where the embryo and endometrium meet. This could help identify specific genes and communication pathways that are essential for a successful pregnancy, lead to new ways to improve IVF success rates, develop biomarkers to assess embryo quality or uterine readiness, to help people achieve successful pregnancies and reduce the heartbreak of early pregnancy loss.

Tissue-resident adult stem cells are medically significant both for their regenerative potential and their vulnerability to drive pathologies, such as cancer, when they acquire the wrong combination of mutations. A rigorous understanding of the normal stem cell is a prerequisite for understanding how it changes in a disease context. Adult brain stem cells are well-studied in mice using experimental methods that can never be done in humans - for example, modifying the genome to label them. However, whether they exist in the human brain - and their relevance in health and disease - is controversial. One problem is that these cells are extremely rare, and so it is essential to have a method for purifying normal human brain stem cells for characterization and experimentation. Our lab has strong preliminary data suggesting that we have captured adult neural stem cells from fresh human brain tissue, and furthermore, that we can purify them alive. We are very close to having enough data for a publication describing these candidate adult human brain stem cells. We have also collected evidence that this cell type plays a role in a least two forms of brain disease involving abnormal tissue growth: malignant glioma, and a nonmalignant form of epilepsy with local regions of brain overgrowth. We wish to systematically follow up on these preliminary observations, as it will set the stage for future studies investigating why and how normal brain stem cells become co-opted in disease. Achieving the objectives described in this proposal will offer a new strategy for studying stem cells in the adult human brain, and boost discovery of new precision therapies for brain tumors and cortical overgrowth epilepsies.

A healthy lymphatic system is vital for keeping our organs functioning properly. When it doesn’t work, it can lead to serious illnesses that affect up to 250 million people worldwide — and currently, there is no cure. Progress has been slow because scientists lack reliable lab models of human lymphatic development and access to patient-specific lymphatic cells. This project addresses both issues by developing a new stem cell-based system to create human lymphatic endothelial cells, offering a powerful tool to understand how these diseases arise and to develop potential treatments. Until now, researchers have mostly relied on animal models to study lymphatic diseases, but many human conditions can’t be accurately mimicked in animals. Our new model gives us, for the first time, a way to study human lymphatic development and disease in a dish.

First, I have developed a method to generate lymphatic cells from human pluripotent stem cells (hPSCs) in a dish at high purity in just 8 days. Next, I will guide these cells to develop into all the lymphatic subtypes found in the body. Using the model, I will study how genetic mutations linked to lymphedema affect each stage of lymphatic cell development.  Early results using stem cells from patients with congenital heart disease suggest that they are more likely to generate dysfunctional lymphatic cells, and I will use this model to explore the underlying reasons. Finally, I aim to develop a method for transplanting these stem cell-derived lymphatic cells into mice to help form a functional lymphatic structure in vivo.

To summarize, developing a human model for how lymphatic cells develop will give us powerful tools to uncover the causes of lymphatic diseases and pave the way for developing targeted and cell-based therapies for lymphatic disorders.

Hematopoietic stem cells (HSCs) are rare cells primarily located in the bone marrow. They are the only cells capable of both self-renewing and producing all types of mature blood cells throughout life. Over the past few decades, studies using transplantation of HSCs into irradiated mice have helped map out how blood cells develop. However, we still don’t fully understand where exactly in the bone marrow these HSCs reside or what types of neighboring cells interact with them.

One major reason for this knowledge gap is the lack of a specific marker that can uniquely indicate HSCs in bone marrow. Without this, it's been difficult to study how HSCs interact with their surrounding environment. Our lab has previously found Hoxb5 gene is expressed only in long-term HSCs and not in other bone marrow cells. Using this discovery, I developed a genetically modified mouse that uses an enhanced fluorescent reporter to clearly label long-term HSCs.

Imaging the bone marrow of these mice revealed that long-term HSCs exist as single cells positioned near blood vessels. Building on this model, I propose to use an advanced spatial transcriptomics technique—which can analyze bone marrow tissue slices 40–50 μm thick (equivalent to at least 4–5 cell layers)—to identify the exact cells that surround and physically contact each long-term HSC in its natural environment. This approach will generate a detailed cellular map of the HSC niche, along with transcriptional profiles of these cells under normal conditions. I will also apply the same method to these reporter mice under bone marrow stress conditions (such as injury or disease) to determine how the surrounding microenvironment changes at the cellular and molecular levels.

The results of this study will reveal, for the first time, the full set of neighboring cells that interact with long-term HSCs and how these cells are affected by bone marrow stress. This knowledge will enhance our understanding of how the HSC microenvironment regulates key behaviors of long-term HSCs, such as dormancy, activation, and migration. Ultimately, these insights could lead to improved strategies for growing and expanding HSCs in the lab for medical applications, and advance therapies involving stem cells and blood-related diseases.

Friedreich ataxia (FRDA) is a rare inherited disease that causes progressive damage to the nervous system and heart. It is caused by a mutation in the FXN gene, where a segment of DNA is abnormally expanded. This mutation reduces the production of frataxin, a protein that helps cells maintain healthy mitochondria. Without enough frataxin, cells, especially in the brain and heart, malfunction over time. There is currently no cure, and available treatments only help manage symptoms.

 In earlier work, we showed that transplanting bone marrow stem cells into FRDA mice helped improve their health. These donor cells could replace immune cells in the heart and brain known as macrophages and microglia the  and deliver healthy mitochondria to damaged brain and heart cells. In this new project, we aim to take that a step further by using the patient’s own stem cells, correcting the genetic mutation using a highly precise technique called prime editing. This method can remove the abnormal DNA repeats without cutting the DNA, making it safer and more accurate. We will test this approach first in mouse and human cells in the lab, and then in a mouse model of FRDA to see if the corrected cells can restore mitochondrial function and improve brain and heart health.

Our ultimate goal is to develop a new, personalized treatment that could help not only people with FRDA, but also those with other mitochondrial diseases caused by similar genetic mutations.

Clonal hematopoiesis of indeterminate potential (CHIP) is a clinically significant aging process in which hematopoietic stem cells (HSC) undergo clonal expansion driven by somatic mutations which confer a proliferative advantage.  CHIP has been associated with an increased risk of hematological cancers, as well as more common aging pathologies such as heart disease.   One of the most effective methods for better understanding the biology of CHIP is through genome wide association studies (GWAS), which have identified variants near genes like TERT as increasing risk of developing CHIP.  Much less is known about what affects how quickly these clones expand, and understanding this is important to develop treatments to slow this expansion and ultimately reduce the disease risks associated with CHIP.  Our lab has developed a method, PACER, for estimating clonal expansion rate in subjects CHIP only from whole genome sequencing data, and a GWAS for PACER identified variants near TCL1A as associated with PACER.  In this proposal, my first aim is to greatly increase the sample size of the GWAS for PACER by a factor of at least seven, so we can discover more risk genes for PACER.  

An additional challenge in the field is better understand clonal hematopoiesis with an unknown driver (CH-UD), which unlike CHIP, does not have a known driver mutation.  Several studies have tried to identify CH-UD and study it with GWAS, but have lacked a rigorous definition of CH-UD or sufficient sample size to identify many loci.  I propose in this study to rigorously define CH-UD using information gained from PACER in CHIP subjects, and to perform a large, well-powered GWAS to identify risk genes for developing CH-UD and for clonal expansion rate in CH-UD.  I will use cutting edge tools from statistical genetics to integrate all of the GWAS I will run with molecular QTL studies and other GWAS to better identify relevant target genes.  Ultimately the target genes identified by these studies will make strong candidates for experimental studies in HSC and mouse models that will ultimately lead to new treatments to slow or prevent clonal hematopoiesis.