Research

The Crane lab is actively involved in research centered on human diseases and disorders. We develop new tools and techniques to answer our hypotheses on mitigating inflammatory responses using cell and animal models. We mainly focus on induced pluripotent stem cells (iPSCs), which have revolutionized the fields of developmental biology and regenerative medicine. The Crane Lab, in collaboration with University of Minnesota faculty, is leveraging iPSCs in the following ways:

Targeted differentiation of iPSCs towards myeloid lineage cells, including macrophages and microglia.

iPSCs have the unique potential to differentiate into any cell type within the body, given the proper signalling cues and culture conditions. Given that many macrophages are present in tissues throughout the body and play an integral role in the pathogenesis of many diseases and disorders, we have established a robust differentiation protocol to generate myeloid lineage cells (macrophages/microglia) from iPSCs that can be used for downstream cellular therapies and disease modelling.

Genome engineering of iPSCs using non-viral transgene insertion and next-generation genome editors, including base editors.

To disrupt gene expression, the CRISPR/Cas9 system has proven to be the most robust. However, next-generation genome editors (base-editors/prime editors) have been developed to precisely alter the genetic code without the need for double-strand breaks. In my lab, we leverage base editor technology in iPSCs to simultaneously disrupt the expression of up to 10 genes.

There are several tools commonly used to insert a gene of interest into the DNA of mammalian cells. Our lab has optimized the design and engineering pipeline using non-viral TcBuster transposon vectors to integrate our transgenes into iPSCs. This system is highly adaptable and has been used to integrate a chimeric antigen receptor (CAR) or logic-gated biosensors, with robust expression of the transgene throughout the differentiation process. We have also developed the tools for targeted transgene knock-in using the CRISPR/Cas9 system.

Using engineered iPSC-derived macrophages to combat inflammation in neurodegenerative diseases and promoting an anti-tumor response is solid tumors.

Macrophages are innate immune cells found in nearly all tissue systems and have basic functions of immune surveillance and tissue homeostasis. Macrophages are important for tissue repair and initiating a hyperactive immune response after detecting pathogens and for this reason have a spectrum of responses that can be dysfunctional in disease. Tumor cells often co-opt the basic functions of macrophages to inhibit the anti-tumor response of the immune system, promote tissue remodeling and vascularization leading to metastasis. Our goal is to develop cellular therapies that do not respond to the tumor microenvironment. Using base editors, we knockout genes involved in these pathways and insert a chimeric antigen receptor in iPSCs, followed by differentiation to macrophages for direct tumor targeting and killing.

In diseases of chronic inflammation, such as inflammatory bowel disease or neurodegenerative disorders, tissue resident macrophages often promote inflammation in a feed-forward cycle. We leverage our genome engineering tools in iPSCs to knockout the expression of genes in these pathways and use logic-gated biosensors to release anti-inflammatory biologics. Differentiation of these engineered cells into macrophages or microglia can then be delivered as a disease modifying therapy.

Our laboratory is dedicated to applying the CRISPR-mediated genome-editing techniques for Gene Therapy and Cancer Research. 
Our long-term goal is to harness the capabilities of CRISPR to develop precise and targeted genome-editing strategies tailored to the challenges of lung and liver disease. 

1. CRISPR Genome Editing Technologies 

The continuous development of CRISPR-Cas genome editing tools is driving significant advances in the life sciences. Currently, four major classes of CRISPR-Cas-derived genome editing agents—nucleases, base editors, transposases/recombinases, and prime editors—enable precise genome modifications in experimental systems, with some rapidly advancing into clinical applications. Each class exhibits unique capabilities and constraints. Our lab has contributed to expanding their editing potential, broadening their target range, and enhancing editing specificity.  

2. in vivo CRISPR off-target analysis 

CRISPR-based genome editing holds great promise for therapeutic applications. Before genome-editing tools can be applied in patients for disease mitigation, an assessment and optimization of their genomic safety is required, with the goal of minimizing unintended editing at off-target sites within the genome. We developed PE-tag technology and GUIDE-tag technology for identification of toxicity and specificity of CRISPR-based genomic medicine. GUIDE-tag enables one-step, unbiased off-target genome editing analysis in mouse liver and lung. Importantly, GUIDE-tag enables oncogene insertion to mouse lung or liver to build mouse models, which will facilitate cancer research. PE-tag enables genome-wide profiling of off-targets from prime editing in vitro using extracted genomic DNA, in mammalian cultured cell lines and in vivo in adult mouse liver. The PE-tag also offers the opportunity to perform off-target analysis directly on patient samples, providing a personalized assessment of genome-wide specificity. In summary, our off-target detection approaches provide platforms for the characterization and optimization of the specificity of CRISPR system in clinical trials.

3. in vivo CRISPR delivery using AAV and LNP

Genome editing in the lung has the potential to provide long-term expression of therapeutic protein to treat lung genetic diseases. Yet efficient delivery of CRISPR to the lung remains a challenge. We developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing of a lox-stop-lox-Tomato reporter in mouse lung airway. We observed ∼19%–26% Tomato-positive cells in both large and small airways, including club and ciliated epithelial cell types. We further showed that intratracheal installation of CRISPR/Cas9 in AAV5 can edit a housekeeping gene or a disease-related gene in the lungs of young rhesus monkeys. We observed up to 8% editing of angiotensin-converting enzyme 2 (ACE2) in lung lobes after single-dose administration. Single-nuclear RNA sequencing revealed that AAV5 transduces multiple cell types in the caudal lung lobes, including alveolar cells, macrophages, fibroblasts, endothelial cells, and B cells. In addition, leveraging a high-throughput platform, we synthesize and screen a combinatorial library of biodegradable ionizable lipids to build inhalable delivery vehicles for messenger RNA and CRISPR–Cas9 gene editors. Lead lipid nanoparticles are amenable for repeated intratracheal dosing and could achieve efficient gene editing in lung epithelium, providing avenues for gene therapy of congenital lung diseases. These highly effective delivery platforms will facilitate the study of therapeutic genome editing in the lung and other tissue types.

4. Pathogenic allele correction and cancer modelling using prime editor

Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. However, the efficacy of prime editing in adult mice has not been established. We developed an NLS-optimized prime editor that improves genome editing efficiency. Using those platforms, we could seed tumor formation through somatic cell editing in the adult mouse. We successfully utilized dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and our findings further established the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction. By delivering those platforms with two adeno-associated virus (AAV) vectors, we could correct the disease-causing mutation in a mouse model of type I tyrosinemia, and could seed tumor formation through somatic cell editing in the adult mouse. Our findings further established the broad potential of this genome editing technology for the direct installation of sequence modifications in vivo, with important implications for cancer modeling and disease correction.

5. Identify drug resistant mechanisms in lung cancer using CRISPR screens

Oncogenic activation of KRAS and the FGFR amplification is frequent in lung and other cancers. However, due to drug resistance, pharmacological blockage of aberrant KRAS or FGFR signaling has provided little clinical benefit in patients. The determining factors for the limited efficacy remain incompletely understood. To identify genes that modulate sensitivity to targeted therapy, we performed kinome-scale CRISPR-Cas9 loss-of-function screens in KRAS-mutant lung cancer cell lines, FGFGR1-amplified squamous cell lines and malignant pleural mesothelioma cell lines. We identified PLK1 as a potent synthetic lethal target that mediates a resistance mechanism by overriding DNA damage and cell cycle arrest upon FGFR1 inhibition. We found that hyperactive mTOR signaling is a characteristic feature of chemoresistance in KRAS-mutant lung cancers. Coherently, combined treatment with mTOR inhibition and chemotherapy synergistically suppressed tumor growth in preclinical mouse models.

Song’s lab focuses on the molecular mechanisms of non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) and their immunotherapies. There are four main areas of focus for Song’s lab:

1. The underlying mechanism by which NAFLD progresses to HCC.
2. Roles of Kupffer cells in modulating the pathogenesis of NAFLD and HCC.
3. MicroRNA-based immunotherapy for the treatment of NAFLD and HCC.
4. Regulation of microRNA biogenesis in NAFLD and HCC.

Current Research Direction

The underlying mechanism by which NAFLD links to HCC. Hepatocellular carcinoma (HCC) is a highly fatal disease with mortality that runs almost parallel to its incidence. The incidence of HCC that was once considered a rare cancer is increasing dramatically in the US. However, it is estimated that 15-50% of HCC patients develop HCC in the absence of eminent etiological factors such as hepatitis viral infection in the Western world. One major cause of this rising incidence is non-alcoholic fatty liver disease (NAFLD). NAFLD progresses to nonalcoholic steatohepatitis (NASH), which increases the risk for the development of liver fibrosis, cirrhosis, and HCC. The incidence rate of NAFLD has been rapidly increasing due to the prevalence of obesity. Despite a strong association between NAFLD and HCC, the underlying mechanisms by which NAFLD patients develop HCC are not known. We are using single-cell RNA-seq, spatial transcriptomic approach, novel mouse models and molecular and cellular approaches to investigate the roles of microRNAs in linking NAFLD to HCC.  

MicroRNA-based immunotherapy of liver cancer. Despite the impressive response of immunotherapies in some cancers, HCC patients have not benefited from immunotherapies. One major reason is that the liver has higher physiological immune tolerance to antigens. Another consideration is that multiple pathways participate in HCC development, establishing a refractory immune tolerogenic tumor microenvironment (TME), thereby leading to resistance to single immunotherapy. MicroRNAs (miRNAs), a class of naturally-occurring small non-coding RNAs, function by simultaneously fine tuning multiple pathways. During the evolution, a stable interactome between miRNAs and their targets is formed to maintain the physiological homeostasis. These characteristics of miRNAs allow us to speculate that miRNAs might be able to precisely modulate immune homeostasis and potentially overcome limitations and challenges of current immunotherapies. We are identifying miRNAs that are critical regulators of immune homeostasis during the pathogenesis of NAFLD and HCC, in addition to developing miRNAs as a novel, effective and accurate immunotherapy without adverse effects that are inherent in the routine immunotherapies.

Roles of Kupffer cells (KCs) in preventing immune escape during the pathogenesis of HCC. KCs, liver resident macrophages, account for ~15% of the liver cell population. KCs represent a major source of chemoattractant molecules, indicating that KCs might be involved in maintaining immune homeostasis. However, their role in HCC is rarely described. We have established that KCs are a critical therapeutic target for the treatment of HCC. We are determining the response of KCs by analyzing secretome and transcriptome of KCs during the pathogenesis of HCC, establishing the interact network between KCs and other immune cells, and finally elucidating the underlying mechanism of immune escape in HCC. Our findings will fill the knowledge gap regarding the role of KCs in preventing immunosuppression and elucidate how KCs prevent immune escape and HCC development. By elucidating the mechanisms involved, we intend to develop KCs as a new therapeutic target against HCC.

Elucidating how hepatic insulin resistance simultaneously drives hepatosteatosis and hyperglycemia. In normal livers, insulin drives lipogenesis and inhibits glucose production. Insulin resistance is the major cause of hepatosteatosis and hyperglycemia. However, the paradox of selective hepatic insulin resistance, wherein the insulin-resistant liver fails to suppress glucose production but continues to produce triglycerides, has been central to the pathophysiology of type 2-diabetes. Our goal is to resolve this long-standing mystery.

Synergistic role of microRNAs and their host genes in maintaining lipid homeostasis in NAFLD. miRNA genes are categorized based on genomic location including, intronic and exonic miRNAs in non-coding transcripts and intronic and exonic miRNAs in protein-coding transcripts. It is widely-accepted that both exonic and intronic miRNAs in protein-coding transcripts share the transcription machinery with their host genes. One such miRNA is miR-378 that is embedded within the first intron of Ppargc1β. Ppargc1β encodes PGC1β, a transcriptional coactivator that regulates mitochondrial biogenesis, thermogenesis, and lipid metabolism. Liver-specific ablation of Ppargc1β leads to reduced FAO and VLDL secretion and subsequently hepatosteatosis. However, it is unknown, how PGC1β and miR-378, which are located in the identical Ppargc1β-miR-378 genomic locus, synergistically and coordinately modulate hepatosteatosis, inflammation and fibrosis. Completion of this study will provide a novel insight into the transcription mechanism(s) of miRNAs and their host genes, which maximizes the genetic information to fit the complexity of NAFLD pathogenesis.
 

Steer’s laboratory has been involved in four major areas of research during the past ten years.

1. The Sleeping Beauty (SB) transposon system functions via a cut-and-paste mechanism catalyzed by the binding of SB transposase to inverted repeats/direct repeats (IR/DRs) of the mariner transposon. It excises the relevant transgene from the transposon at the IR/DRs and inserts the element into random TA dinucleotide sites within the genome. They are applying SB as a gene therapy vector to a variety of different animal disease models, including liver, bone marrow and brain disorders. Steer's laboratory is also interested in characterizing the effects of SB transposition on genomic methylation and histone acetylation.
2. Using ursodeoxycholic acid (UDCA), a hydrophilic bile acid, as a potent antiapoptotic agent. They have used UDCA as a therapeutic agent to treat transgenic models of Huntington’s disease and retinitis pigmentosa as well as acute stroke, spinal cord injury, myocardial infarction, and acute renal failure. Steer's laboratory continues to study basic mechanisms and translational applications of UDCA; and how it functions to maintain cell survival.
3. Characterizing the role of microRNAs in gene regulation for a number of different target organs and stem cell populations. The Steer lab has identified specific microRNAs that may be involved in the progression of colon from polyp to cancer, as well as their role in the regenerating liver. The studies are both basic and translational in nature. They are also identifying specific microRNAs as biomarkers of disease that can be assayed in blood. Most notably, they have discovered a unique nuclear profile of mature microRNAs; and a subset of microRNAs in mitochondria that may act as a rheostat for the control of apoptosis.
4. The major focus of Steer’s lab is now focused on regenerative medicine, and research to create human livers in pigs for transplantation. The approach utilizes a combination of gene editing and a technology referred to as blastocyst complementation. The ultimate goal is to create human livers in pigs that are immunology identical to the recipient, thereby not requiring the use of immunosuppression…a paradigm shift in organ transplantation.  They have received a 5-year NIH R01 to carry out the studies. In brief, there are more than 20,000 patients waiting to receive liver transplants. The number is increasing due to an aging US population accompanied by an increasing incidence of chronic liver diseases associated with such disorders as alcoholic liver disease, hepatitis, MAFLD and NASH. The demand increasingly outstrips the supply for organ transplantation. One solution to this problem is the ability to generate human livers in animals for liver as well as hepatocyte transplantation. It is now possible to regenerate the replica of organs/cells from one species of animal within the body of a second species. This involves the knockout (KO) of specific developmental genes in the blastocyst of species two; and the intra-blastocyst injection of pluripotent stem cells from species one to generate offspring that carry organs/cell types derived from that donor. The translation of this approach requires an efficient gene-editing technology. As preliminary data, the Steer lab hypothesized that rat liver can be generated in the mouse by the injection of rat ESCs into CRISPR-genetically engineered murine blastocysts and transplanted back into syngeneic rats. The studies represent a first step of interspecies development of exogenic organs for transplantation without immunosuppression. They are designed to characterize (1) intra- and interspecies exogenic liver and endothelium derived from HHEX KO embryos; (2) the immunology and function of interspecies exogenic liver and endothelial development derived from HHEX KO embryos; and (3) several approaches to enhance the generation of interspecies chimeras that include humanization of morphogen ligand-receptor interactions. The resulting exogenic rat liver and endothelium is transplanted back into syngeneic rats to evaluate graft survival and functionality. The generation of whole livers that are comprised primarily of rat hepatic and endothelial cells derived from implanted rat ESCs would represent a paradigm shift and provide the necessary preclinical evidence for ultimately creating human livers in animals.

Co-directed by Dr. Byron Vaughn and Dr. Eugenia Shmidt, the Inflammatory Bowel Disease (IBD) Program focuses on improving care and finding a cure for IBD. 

The team studies how to use and develop microbiota-based treatments to prevent and manage IBD. They also research how different therapies—both traditional and nutrition-based—affect the gut microbiome.

Another key area is improving current treatments through drug level monitoring. The program also studies IBD during pregnancy, including the safety of medications and how to manage disease throughout pregnancy.

The program offers industry-sponsored research, including phase III and IV clinical trials for different types of IBD.

View our Active Clinical Trials

The UMN Esophageal Disorders Program is led by Dr. Joshua Sloan. Dr. Sloan’s research interests include Eosinophilic Esophagitis, esophageal motility disorders, Esophageal Lichen Planus, and Functional Lumen Imaging Probe among other topics.

Dr. Sloan is currently active on a few different research projects, including:

• Ongoing retrospective multicenter study focusing on Esophageal Lichen Planus
• Clinical trial on Eosinophilic Esophagitis
• Esophageal motility in the setting of lung transplant patients
• Retrospective eosinophilic esophagitis projects
• Retrospective analysis evaluating patients with Fanconi Anemia

Beyond Dr. Sloan’s current research work, the UMN Esophageal Disorders Program is interested in collaborating with dieticians and GI clinical psychologists to evaluate how their involvement in patient care may impact outcomes

Led by Dr. Alex Khoruts, The University of Minnesota Microbiota Therapeutics Program pioneered the use of Intestinal Microbiota Transplantation (IMT) to restore intestinal health and created the first stool donor program worldwide. There are many potential clinical applications of microbiota therapeutics. Our program is committed to support academic trials for a variety of applications. Some of the ongoing work is in the areas of ulcerative colitis, leukemia, pitt hopkins syndrome, autism, advanced liver disease, and others.

Dr. Thomas Leventhal is an advanced liver disease and critical care expert. Dr. Leventhal's research interests include acute liver failure, critical care for those with advanced liver disease, organ donor management, and generic drug costs. Dr. Leventhal is currently active working on the following research projects, including

• Phase 1 trial of a novel device of a bio-artificial liver to provide support to those with acute liver failure
• Cost of care and pharmacotherapy - including membership in a Drug Cost consortium studying the costs of generic medications in the United States and studying financial toxicity of commonly used medications in patients with cirrhosis
• Outcomes of patients with cirrhosis admitted to the intensive care unit

Financial Burden and Cost of Care
Dr. Elizabeth Aby is conducting prospective, multi-center research evaluating the impact of financial burden and cost of care on patients with chronic liver disease who are undergoing liver transplant evaluation.

Primary Sclerosing Cholangitis
Drs. Elizabeth Aby & Jack Lake are collaborating with Dr. Alex Khoruts to perform a pilot study to evaluate the initial safety and feasibility of microbiota transplant therapy (MTT) in adult patients with primary sclerosing cholangitis (PSC). 

The majority of the Division's funded clinical research is in the area of viral hepatitis. There are currently four funded research protocols for the management of patients with hepatitis C. Dr. John Lake is involved in clinical research studies which focus on the treatment of hepatitis C with pegylated interferon and ribavirin.

In addition, the Division is an active participant in the Midwest Hepatitis Study Group, which is a consortium of investigators interested in the management of patients with chronic viral hepatitis. Dr. John Lake is also a co-investigator on 2 NIH-funded clinical research protocols, one on the area of primary biliary cirrhosis (M. Eric Gershwin, PI) and one in the area of non-alcoholic steatohepatitis (Elizabeth Parks, PI).

The Gastroenterology Division maintains an ongoing interest in the use of TIPS (transjugular intrahepatic portal systemic shunt) for management of complications of portal hypertension.

Led by Dr. Guru Trikudanathan, MBBS, the section is active in research focusing on diabetes and pancreatitis and genetic factors that predispose to pancreatitis or diabetes that affect TPIAT outcomes. Listed below are current studies:

• Dr. Guru Trikudanthan is the Co-Principal investigator at University of Minnesota for Diabetes Related to Acute pancreatitis and its Mechanisms (DREAM) and The IMaging Morphology of Pancreas in DIabetic PatieNts following AcutE PaNcreaTitis (IMMINENT) study from NIH/NIDDK as a part of the Type 1 Diabetes in Acute Pancreatitis  Consortium (T1APDC) UO1. DREAM study is a prospective longitudinal observational clinical study to investigate the incidence, etiology and pathophysiology of diabetes mellitus following acute pancreatitis. A biorepository of longitudinally collected samples from participants will be developed for use in future translational studies. IMMINENT study aims to assess morphologic changes on MRI as a imaging biomarker to predict who will develop diabetes following acute pancreatitis.
• Dr. Guru Trikudanthan is also the principal investigator of GEN POST study which is funded by the American Pancreas Association Young Investigator award. Gen POST capitalizes on POST (the Prospective Observational Study of Total pancreatectomy and islet autotransplantation) study infrastructure funded by NIDDK, to address specifically how outcomes after TPIAT are determined by genetic factors that predispose to pancreatitis or diabetes or that affect opioid responsiveness. GEN POST hopes to predict how genotype affects phenotypic response to TPIAT.
• Dr. Guru Trikudanathan is also the coinvestigator at University of Minnesota for PROCEED study from NIH/NIDDK as part of the Consortium for the Study of Chronic Pancreatitis, Diabetes and Pancreatic Cancer (CPDPC) (UO1). PROCEED is the first prospective observational cohort study for chronic pancreatitis in the United States which aims to define disease progression and develop a platform to conduct translation and mechanistic studies in chronic pancreatitis. This study will enroll adults at different stages of chronic pancreatitis and provide the most accurate and reliable estimate to date on progression of chronic pancreatitis. The established cohort and biorepository will also facilitate development of new strategies for diagnosis, methods to monitor disease progression and treatment of chronic pancreatitis.

ERCP research in the section is led by Dr. Martin Freeman, MD. Dr. Freeman has published over 30 textbook chapters and 250 peer reviewed articles, including several index papers for which he was the principal author: a multicenter study on ERCP complications in the New England Journal of Medicine, a multivariable model for prediction of post ERCP pancreatitis in Gastrointestinal Endoscopy, guidelines for the management of acute pancreatitis in the American Journal of Gastroenterology, and a consensus paper and a systematic review on interventions for necrotizing pancreatitis in the Pancreas, and recently in Gastroenterology.

Through work led by Dr. Stuart Amateau MD, PhD, the advanced endoscopy section has collaborated with regional and national partners to develop novel devices and techniques to improve patient care. Specific examples of innovation include a refined approach to malignant obstruction of the bile duct that is not accessible by traditional endoscopic retrograde cholangiopancreatography ERCP and development of a novel means to deploy hemostatic particles for the treatment of gastrointestinal bleeding using considerably less expensive components.

Led by Dr. Nabeel Azeem, MD, the section is active in research on endoscopic resection of gastrointestinal neoplasia, endoscopic therapy for achalasia, third space endoscopy, and EUS guided treatment of gastric and ectopic varices.

Led by Dr. Shawn Mallery, MD, the advanced endoscopy section is active in research aimed at discovering the effectiveness and use cases of interventional EUS to improve patient diagnosis and reduce the need for traumatic surgery. Of note, recent publications have:

• evaluated the impact of telepathology and operator experience on endobronchial ultrasound- and endoscopic ultrasound-guided fine-needle aspiration (EBUS-/EUS-FNA)
• measured the effectiveness of EUS-guided pancreaticogastrostomy (PG) as an alternative to surgery to drain ducts for treatment of disconnected pancreatic duct syndrome (DPDS)
• described the clinical, radiologic, cytologic, gross and histologic features of diagnosing mediastinal ganglioneuroma by transesophageal endoscopic ultrasound guided fine needle aspiration (EUS-FNA)
• discovered a potential key role for fine-needle aspiration cytology in the preoperative diagnosis of pancreatic and other intra-abdominal desmoid tumors

The section’s faculty are active in research on the early diagnosis and management of chronic pancreatitis and the management of and interventions for necrotizing pancreatitis. In addition, studies are ongoing with a focus on biliary disease, obstruction, strictures, and leaks. These studies highlight treatments and procedures including ERCP, stents, and lithotripsy devices. Research is also focused on pancreatic endo-therapy through endoscopic ultrasound and endoscopic retrograde cholangio-pancreatography for management of complex pancreatic and biliary conditions.

COVID-19 & Acute Pancreatitis. The advanced endoscopy section was the first to publish a significant increase in both the morbidity and mortality of patients with acute pancreatitis that were also found to have evidence of SARS-CoV-2 infection when compared to an uninfected cohort. The study demonstrated a higher incidence of multiorgan failure and persistent organ failure in the infected group prompting further investigations into a relationship with the inflammatory cascade.

Total Pancreatectomy and Islet Autotransplantation. The section has recently studied the effects that preoperative sarcopenia has on islet cell yield following TPIAT. In addition, studies have been conducted on the alterations in enteroendocrine hormones, reduced bone mineral density, and long term diabetes outcomes after TPIAT.