The Sixth Annual Next Gen

Stem Cell Conference

When: August 2-3, 2018

Where: West Hartford, CT

Advanced registration ends: June 29, 2018

Speaker Information

Life-saving regeneration of the entire human epidermis by transgenic stem cells

Laura De Rosa, Ph.D.
Keynote Speaker 

Center for Regenerative Medicine ‘Steffano Ferrari’
University of Modena and Reggio Emilia, Italy

Bio: Laura De Rosa earned her PhD at University of Naples in 2009. From 2009 to 2011 she was a PostDoc at Caterina Missero Lab. Since 2011 she is senior Postdoc at the Centre for Regenerative Medicine “S. Ferrari” in the group of Prof. Michele De Luca, leading scientist in the field of stem cell translational medicine.

Abstract:
Epidermal stem cells (EpSCs) are an attractive cell type for autologous gene correction and have been shown to be amenable to such techniques. Junctional Epidermolysis Bullosa (JEB) LAMB3 dependent was the first genetic disease targeted by transplantation of autologous epidermal cultures originated from genetically EpSC. Mavilio et al. and Bauer et al. provide compelling evidence that local transplantation of transgenic epidermal sheets can regenerate a functional epidermis, leading to permanent and safe correction of skin lesions in JEB patients. This evidence was confirmed by our work providing proof for skin regeneration of the over 80% of the body surface in a 7-year-old JEB patient from gene corrected EpSCc. Provirus integration analysis conducted in this study revealed that most of the integrations occurred in non-protein-coding sequences. The genes that contained integrated retroviral vectors are not known to be directly involved in cancer-related processes, suggesting the safety of the approach. In addition, the similar integration patterns observed in vitro and in vivo showed that neither the culture protocol nor natural skin cell turnover led to preferential survival and expansion of particular cell clones. Clonal tracing conducted in this work showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study not only proved the feasibility but also confirmed the safety of a combined stem cell and gene replacement therapy for the treatment of this divesting rare disease, diminishing the unceasing doubts on the use of MLV-RV vectors and the genotoxic risk associated with their uncontrolled insertion into the genome, reported with hematopoietic stem cells, providing a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

Micropatterning hair follicles and vasculature in human skin constructs using 3D-printing

H. Erbil Abaci, Ph.D.
Assistant Professor
Department of Dermatology,
Columbia University Medical Center

Abstract:
Human skin constructs (HSCs) have the potential to provide an effective therapy for patients with significant skin injuries and to enable human-relevant drug screening. However, incorporation of engineered skin appendages, such as hair follicles (HFs), into HSCs has been a long-standing challenge. Employing 3D-printing technology, we initially developed a biomimetic approach for generation of human HFs within HSCs through guiding physiological 3D organization of cells in the HF microenvironment. Our method allows for restoration of dermal papilla cell (DPC) hair inductivity by controllable DPC spheroid formation and initiation of the crosstalk between epidermal-mesenchymal cells, leading to HF induction in HSCs in an entire ex vivo context. However, engraftment of these HSCs onto immunodeficient mice did not efficiently induce de novo hair formation; instead, we found necrosis at the center of the grafts. Histological analyses showed that there was a lack of vasculature and blood supply in the grafts. To address this, we prevascularized the HSCs containing 255 HFs/cm2 by encapsulating GFP-tagged human umbilical vein endothelial cells (HUVECs) into the dermal compartment. The stimulation of HUVECs with endothelial growth factors induced spontaneous capillary formation in vitro, where the capillary networks surrounded the HFs. Engraftment of prevascularized HSCs onto immunodeficient mice led to human hair growth, confirmed by human-specific nuclear staining and PCR analyses using human vs. mouse-specific primers. We also found that HUVECs in the grafts formed an organized capillary network with the surrounding mouse vessels and promoted blood supply to the grafted skin/hair. The ability to regenerate an entire HF from cultured human cells will have a transformative impact on the medical management of different types of alopecia. This strategy may provide a skin replacement therapy for patients with significant skin and hair loss.

Modeling ALS vulnerability with ESC-derived cranial and spinal motor neurons

Disi An
Ph.D. Student
Biology Department of New York University

Abstract:
Human skin constructs (HSCs) have the potential to provide an effective therapy for patients with significant skin injuries and to enable human-relevant drug screening. However, incorporation of engineered skin appendages, such as hair follicles (HFs), into HSCs has been a long-standing challenge. Employing 3D-printing technology, we initially developed a biomimetic approach for generation of human HFs within HSCs through guiding physiological 3D organization of cells in the HF microenvironment. Our method allows for restoration of dermal papilla cell (DPC) hair inductivity by controllable DPC spheroid formation and initiation of the crosstalk between epidermal-mesenchymal cells, leading to HF induction in HSCs in an entire ex vivo context. However, engraftment of these HSCs onto immunodeficient mice did not efficiently induce de novo hair formation; instead, we found necrosis at the center of the grafts. Histological analyses showed that there was a lack of vasculature and blood supply in the grafts. To address this, we prevascularized the HSCs containing 255 HFs/cm2 by encapsulating GFP-tagged human umbilical vein endothelial cells (HUVECs) into the dermal compartment. The stimulation of HUVECs with endothelial growth factors induced spontaneous capillary formation in vitro, where the capillary networks surrounded the HFs. Engraftment of prevascularized HSCs onto immunodeficient mice led to human hair growth, confirmed by human-specific nuclear staining and PCR analyses using human vs. mouse-specific primers. We also found that HUVECs in the grafts formed an organized capillary network with the surrounding mouse vessels and promoted blood supply to the grafted skin/hair. The ability to regenerate an entire HF from cultured human cells will have a transformative impact on the medical management of different types of alopecia. This strategy may provide a skin replacement therapy for patients with significant skin and hair loss.

A microRNA signature and TGF-β1 response were identified as the key master regulators inhibiting stem cell functions for spaceflight response

Afshin Beheshti, Ph.D.
Bioinformatician and Data Analyst
KBRWyle, Space Biosciences Division, NASA Ames Research Center
Assistant Professor
Tufts University School of Medicine

Abstract:
Translating fundamental biological discoveries from NASA Space Biology program into health risk from space flights has been an ongoing challenge. Although determination of risk has been a focus for NASA research, data examining systemic (i.e., multi- or pan-tissue) responses to space flight are sparse. The overall goal of our work is to identify potential “master regulators” responsible for such responses to microgravity conditions. To do this we utilized the NASA GeneLab database which contains a wide array of omics experiments, including data from: 1) different flight conditions (space shuttle (STS) missions vs. International Space Station (ISS); 2) different tissues; and 3) different types of assays that measure epigenetic, transcriptional, and protein expression changes. Systems biology analysis of transcriptomic data from seven different rodent datasets reveals for the first time the existence of potential “master regulators” coordinating a systemic response to microgravity and/or space radiation. The analysis of the 7 datasets contained following tissues: liver, kidney, adrenal gland, thymus, mammary gland, skin, and skeletal muscle (soleus, extensor digitorum longus, tibialis anterior, quadriceps, and gastrocnemius). This unbiased approach identifies the key genes/drivers for each study, with TGF-β1 being the most common regulator. We hypothesized the space environment leads to the release of biomolecules circulating inside the blood stream which target TGF-β1. Datamining using key genes identified in each dataset reveals 13 candidate microRNAs (miRNA) which are common in all studies and directly interact with TGF-β1. Surprisingly, we were also able to predict that these key drivers (both the miRNAs and TGF-β1) lead to immunosuppression and inhibition of stem cell activity due to environment in space. Drugs interacting with circulating miRNAs or master regulators such as TGF-β1 could potentially lead to novel countermeasures for spaceflight side effects and should be further investigated. More generally, this study exemplifies the utility of the GeneLab data repository to aid in the process of performing novel hypothesis–based research.

IP6K1 Regulates Mesenchymal Stem Cell Dependent Osteogenesis and Adipogenesis during Disuse, Disease, and Aging

Sid Boregowda, Ph.D.
Research Associate
Scripps Research Institute

Abstract:
Bone marrow-derived mesenchymal stem cells (MSCs) differentiate into chondrogenic, osteogenic, or adipogenic lineages and support hematopoiesis by forming a unique bone marrow niche in an age-depended manner. MSCs during prenatal and postnatal development of skeleton undergo chondrogenic and osteogenic differentiation. However, during organismal aging their potential shifts increasingly toward adipogenesis at the expense of osteogenesis. Therefore, the balance between osteogenic vs. adipogenic differentiation provides a measure of MSC-dependent bone marrow aging. We demonstrate a similar MSC-dependent aging phenotype of bone marrow using hind limb-unloading model of disuse and high fat diet model of obesity in mice. Based on the role of IP6Ks in regulating cell metabolism and survival we hypothesized that this kinase family may also contribute to stem cell homeostasis. IP6K1 is the major isoform among family of three IP6 kinases (IP6Ks), which synthesize inositol pyrophosphate (IP7) from inositol hexakisphosphates (IP6). Deletion of IP6K1 protects mice from high fat diet induced obesity and insulin resistance. IP6K1 promotes insulin resistance by inhibiting the insulin sensitizing protein kinase AKT. Here we demonstrate that primary mouse MSCs derived from IP6K1 knockout mice display increased growth and survival as evidenced by lower expression of mitochondrial reactive oxygen species and Trp53 protein. Moreover, IP6K1-/- MSCs exhibit enhanced osteogenic differentiation and delayed adipogenic differentiation. Additionally, MSCs isolated from old IP6K1-/- mice are protected from aging phenotype. We identified AEBP1 and ALDH2 as major IP6K1-interactors in MSCs that are involved in stem cell maintenance and differentiation. These studies identify IP6K1 as a novel regulator of MSC fate decision and suggest that targeting the IP6K1 signaling network may offer a new therapeutic strategy to ameliorate bed rest-, diabetes-, and age-related osteoporosis.

Investigate Human Spermatogonial Stem Cells Development and Pluripotency Potentials using Genomic Profiling

Jingtao Guo
Graduate student and Postdoctoral researcher
Howard Hughes Medical Institute and Huntsman Cancer Institute
Department of Oncological Sciences, School of Medicine, University of Utah

Abstract:
Human adult spermatogonial stem cells (hSSCs) are the germline stem cells of adult males and display a set of fascinating stem cell properties. First, they must maintain a germline identity and a paternal-specific pattern of epigenetic imprinting. Second, through communication with their testicular niche, they delicately balance self-renewal with differentiation long-term, to avoid exhaustion and allow lifelong gametogenesis. Third, although they are stem cells, hSSCs are “unipotent,” despite stages of amplification and differentiation, their developmental trajectory culminates in the formation of only one cell type—mature sperm.
To understand those proterties, we profiled DNA methylation and open chromatin (ATAC-seq) in SSEA4+ hSSCs, analyzed bulk and single-cell RNA transcriptomes (RNA-seq) in SSEA4+ hSSCs and differentiating c-KIT+ spermatogonia, and performed validation studies via immunofluorescence. First, DNA hypomethylation at embryonic developmental genes supports their epigenetic “poising” in hSSCs for future/embryonic expression, while core pluripotency genes (OCT4 and NANOG) were transcriptionally and epigenetically repressed. Interestingly, open chromatin in hSSCs was strikingly enriched in binding sites for pioneer factors (NFYA/B, DMRT1, and hormone receptors). Remarkably, single-cell RNA-seq clustering analysis identified at least four cellular/developmental states during hSSC differentiation, involving major transitions in cell-cycle and transcriptional regulators, splicing and signaling factors, and glucose/mitochondria regulators. Overall, our results outline the dynamic chromatin/transcription landscape operating in hSSCs and identify crucial molecular pathways that accompany the transition from quiescence to proliferation and differentiation.

Blood-brain barrier endothelium derived from human iPSCs: development, discovery, and devices

Ethan Lippman, Ph.D.
Assistant Professor
Department of Chemical and Biomolecular Engineering
Vanderbilt University

Abstract:
The blood-brain barrier (BBB), which is comprised of the endothelial cells that line brain capillaries and separate the bloodstream from the brain, maintains brain health and homeostasis. To achieve this purpose, the BBB prevents paracellular diffusion of ions and hydrophilic molecules via robust intercellular junctional connections, suppresses nonspecific pinocytosis/transcytosis activity, utilizes polarized molecular transporters to shuttle molecules to and from the brain, and expresses a cohort of efflux transport proteins that prevent penetrance of small, lipophilic compounds. When intact, the BBB hinders drug delivery to the brain, and in a number of disorders, the BBB becomes dysfunctional in ways that can initiate or exacerbate neurological decline. Unfortunately, the role of BBB in these processes has historically been difficult to study in vitro due to a limited supply of high-fidelity cells. My research group has overcome some of these challenges by using human induced pluripotent stem cells (iPSCs) to generate an unlimited quantity of robust BBB endothelium. In this talk, I will briefly describe: (1) our efforts to make the differentiation process fully defined, which greatly extends its capabilities; (2) how we are using the differentiation process to uncover new mechanisms of BBB regulation; (3) how we are transitioning iPSC-derived BBB endothelium into three-dimensional assembles to generate more complex neurovascular structures for disease modeling and drug screening applications.

Developing safe pancreatic cells for diabetes treatment

Cristina Nostro, Ph.D.
Assistant Professor,
McEwan Center for Regenerative Medicine

Abstract:
The current treatment for type I diabetes is insulin injection. While effective, it is not a perfect substitute to continuous insulin release by one’s own b-cells. Recent improvements and successes in islet transplantation for the treatment of type I diabetes demonstrate that cell therapy could become a reality in the management of this disease. However, the low number of islets obtainable from donors limits their therapeutic use. The potential to generate β-cells from human embryonic and induced pluripotent stem cells (hESCs, hiPSCs) differentiated in vitro offers the possibility of a novel and unlimited source of insulin-producing cells for transplantation for the treatment of this disease.Here, we present our recent work using the pancreatic secretory granule membrane major glycoprotein 2 (GP2) to enrich hESC-derived pancreatic progenitors for the generation of β-like cells (C-PEPTIDE+/NKX6-1+) in vitro and in vivo.

Modeling Disease of the Peripheral Nervous System

Nadja Zeltner, Ph.D.
Assistant Professor,
Center for Molecular Medicine
Department of Biochemistry & Molecular Biology
Department of Cellular Biology
University of Georgia

Abstract:
Functional and molecular aspects of human genetic disease can be recapitulated in vitro using patient-specific pluripotent stem cells (PSCs). Familial Dysautonomia (FD) is a debilitating developmental and degenerative disorder that primarily affects derivatives of the neural crest (NC), such as the peripheral nervous system (PNS). For unknown reasons, FD patients present with mild or severe disease despite carrying the identical, homozygous point mutation in IKBKAP. We present in vitro phenotypes at various stages of development that capture severe and mild FD in human PSC-derived cellular lineages. Patient-specific cells only from severe but not mild FD display an impaired capacity of developing into NC derivatives, such as autonomic and sensory neurons, thus they have neurodevelopmental defects. Interestingly however, both severe and mild FD cells show defects in peripheral neuron survival, indicating neurodegeneration as the primary culprit in mild FD. Importantly, we found that neuronal degeneration in mild FD can be halted by treatment with candidate therapeutic compounds. Genetic rescue of the FD mutation in severe FD iPSCs reversed NC, but not sensory neuron lineage phenotypes, implicating that the known FD mutation does not account for all symptoms. Employing whole-exome sequencing, we identified candidate mutations that were only found in severe but not mild FD patients, providing evidence that FD may constitute two genetic sub-diseases. Our study demonstrates that human iPSC-based disease modeling is sensitive in recapitulating disease severity. This paves the road for applications in personalized medicine and raises the prospect that individual patient's disease could be studied in vitro.
Screening a library of small molecules, we further identified a novel compound that rescued severe FD defects. This compound has two known functions. The first is crosslinking of extracellular matrix proteins. Interestingly the secondary mutation we discovered in FD is in LAMB4, an extracellular matrix protein, prompting us to further investigate the interaction between LAMB4 and this compound. Second, the compound is the active chemical in a Traditional Chinese Medicine, making it an interesting possible treatment option for preventing neurodegeneration in FD and possibly more common peripheral neuropathies.

Liver progenitor cell contribution to liver regeneration

Fatima Rizvi, Ph.D.
Post-Doctoral Fellow
Center for Regenerative Medicine
Boston University School of Medicine and Boston Medical Center

Abstract:
In view of the shortage of liver donors, alternatives for liver transplantation therapy need to be searched. Accelerating intrinsic liver regeneration by triggering expansion and differentiation of facultative liver progenitor cells (LPCs) in vivo could prove to be an effective therapy. The receptor KDR, also known as VEGFR2/FLK1, serves as a marker for a distinct and conserved human and murine endodermal fetal liver progenitor cell. Consistent with the fetal progenitor cell features of LPCs, KDR was found to be expressed by more than 70% of adult LPCs in bile duct ligation model of chronic liver injury in mice. We are presently investigating the molecular heterogeneity of LPCs based on expression for KDR and other known LPC markers, and their ability to proliferate and differentiate into hepatocytes in acute and chronic liver injury mouse models.
The evidence of KDR expression on a specific subset of LPCs may make possible the manipulation of the receptor whose activity can be induced in vivo to trigger expansion and differentiation of LPCs as an alternative to treat acute and chronic liver disease.

Planarian: An in vivo petri dish for studying immunological regulation of tissue repair

Margarita Khariton
Ph.D. Student
Wang Laboratory
Department of Bioengineering
Stanford University

Abstract:
A frontier in stem cell research is the intertwined connections between the immune system and tissue regeneration, which have been extensively studied over recent years. The complicated nature of these systems, however, leaves many unanswered questions regarding the underlying mechanisms. The flatworm planarian, owing to its exceptional regenerative abilities and simplicity in laboratory practice, serves as an ideal system for regeneration studies. For the first time, we have isolated the immune cells of the planarian Schmidtea mediterranea and established their transcriptional profile. We have shown these cells as performing active immunological functions and presenting a number of gene expression signatures implicated in wound healing, inflammation, and, most compelling, tissue patterning. Our results suggest that these cells may play a series of complex functions in response to wound healing: besides the common immunological functions, they also initiate feedback pathways for resolution of inflammation, followed by serving as mediators of tissue repair and reorganization through regenerative cues. The revelation of these mechanisms within the planarian provides an avenue for resolving immunological regulatory mechanisms across species that serve to control tissue reconstruction upon injury.

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