Key factor screening in mouse NASH model using single-cell sequencing combined with machine learning.

Aims: To identify and analyze genes closely related to the progression of nonalcoholic steatohepatitis (NASH) by employing a combination of single-cell RNA sequencing and machine-learning algorithms. Main methods: Single-cell RNA sequencing (scRNA-seq) analysis was performed to find the cell population with the most significant differences between the Chow and NASH groups. This approach was used to validate the developmental trajectory of this cell population and investigate changes in cellular communication and important signaling pathways among these cells. Subsequently, high dimensional Weighted Gene Co-expression Network Analysis (hdWGCNA) was used to find the key modules in NASH. Machine learning analyses were performed to further identify core genes. Deep learning techniques were applied to elucidate the correlation between core genes and immune cells. The accuracy of this correlation was further confirmed using deep learning techniques, specifically Convolutional Neural Networks. Key findings: By comparing scRNA-seq data between the Chow and NASH groups, we have observed a notable distinction existing in the Kupffer cell population. Signaling interactions between hepatic macrophages and other cells were significantly heightened in the NASH group. Through subsequent analysis of macrophage subtypes and key modules, we identified 150 genes tightly associated with NASH. Finally, we highlighted the 16 most significant core genes using multiple iterations of machine learning. Furthermore, we pointed out the close relationship between core genes and immune cells. Significances: Using scRNA-seq analysis and machine learning, we can distinguish NASH-related genes from large genetic datasets, providing theoretical support in finding potential targets for the development of novel therapies.

A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells.

Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells (ECs) that play an important role in liver development and regeneration. Additionally, it is involved in various pathological processes, including steatosis, inflammation, fibrosis and hepatocellular carcinoma. However, the rapid dedifferentiation of LSECs after culture greatly limits their use in vitro modeling for biomedical applications. In this study, we developed a highly efficient protocol to induce LSEC-like cells from human induced pluripotent stem cells (hiPSCs) in only 8 days. Using single-cell transcriptomic analysis, we identified several novel LSEC-specific markers, such as EPAS1, LIFR, and NID1, as well as several previously revealed markers, such as CLEC4M, CLEC1B, CRHBP and FCN3. These LSEC markers are specifically expressed in our LSEC-like cells. Furthermore, hiPSC-derived cells expressed LSEC-specific proteins and exhibited LSEC-related functions, such as the uptake of acetylated low density lipoprotein (ac-LDL) and immune complex endocytosis. Overall, this study confirmed that our novel protocol allowed hiPSCs to rapidly acquire an LSEC-like phenotype and function in vitro. The ability to generate LSECs efficiently and rapidly may help to more precisely mimic liver development and disease progression in a liver-specific multicellular microenvironment, offering new insights into the development of novel therapeutic strategies.

Molecular Pathways Regulating Circadian Rhythm and Associated Diseases.

Circadian rhythms, the natural cycles of physical, mental, and behavioral changes that follow a roughly 24-hour cycle, are known to have a profound effect on the human body. Light plays an important role in the regulation of circadian rhythm in human body. When light from the outside enters the eyes, cones, rods, and specialized retinal ganglion cells receive the light signal and transmit it to the suprachiasmatic nucleus of the hypothalamus. The central rhythm oscillator of the suprachiasmatic nucleus regulates the rhythm oscillator of tissues all over the body. Circadian rhythms, the natural cycles of physical, mental, and behavioral changes that follow a roughly 24-hour cycle, are known to have a profound effect on the human body. As the largest organ in the human body, skin plays an important role in the peripheral circadian rhythm regulation system. Like photoreceptor cells in the retina, melanocytes express opsins. Studies show that melanocytes in the skin are also sensitive to light, allowing the skin to "see" light even without the eyes. Upon receiving light signals, melanocytes in the skin release hormones that maintain homeostasis. This process is called "photoneuroendocrinology", which supports the health effects of light exposure. However, inappropriate light exposure, such as prolonged work in dark environments or exposure to artificial light at night, can disrupt circadian rhythms. Such disruptions are linked to a variety of health issues, emphasizing the need for proper light management in daily life. Conversely, harnessing light's beneficial effects through phototherapy is gaining attention as an adjunctive treatment modality. Despite these advancements, the field of circadian rhythm research still faces several unresolved issues and emerging challenges. One of the most exciting prospects is the use of the skin's photosensitivity to treat diseases. This approach could revolutionize how we think about and manage various health conditions, leveraging the skin's unique ability to respond to light for therapeutic purposes. As research continues to unravel the complexities of circadian rhythms and their impact on health, the potential for innovative treatments and improved wellbeing is immense.

Developing Humanized Animal Models with Transplantable Human iPSC-Derived Cells.

Establishing reliable and reproducible animal models for disease modelling, drug screening and the understanding of disease susceptibility and pathogenesis is critical. However, traditional animal models differ significantly from humans in terms of physiology, immune response, and pathogenesis. As a result, it is difficult to translate laboratory findings into biomedical applications. Although several animal models with human chimeric genes, organs or systems have been developed in the past, their limited engraftment rate and physiological functions are a major obstacle to realize convincing models of humans. The lack of human transplantation resources and insufficient immune tolerance of recipient animals are the main challenges that need to be overcome to generate fully humanized animals. Recent advances in gene editing and pluripotent stem cell-based xenotransplantation technologies offer opportunities to create more accessible human-like models for biomedical research. In this article, we have combined our laboratory expertise to summarize humanized animal models, with a focus on hematopoietic/immune system and liver. We discuss their generation strategies and the potential donor cell sources, with particular attention given to human pluripotent stem cells. In particular, we discuss the advantages, limitations and emerging trends in their clinical and pharmaceutical applications. By providing insights into the current state of humanized animal models and their potential for biomedical applications, this article aims to advance the development of more accurate and reliable animal models for disease modeling and drug screening.

The Values and Perspectives of Organoids in the Field of Metabolic Syndrome.

Metabolic syndrome (MetS) has become a global health problem, and the prevalence of obesity at all stages of life makes MetS research increasingly important and urgent. However, as a comprehensive and complex disease, MetS has lacked more appropriate research models. The advent of organoids provides an opportunity to address this issue. However, it should be noted that organoids are still in their infancy. The main drawbacks are a lack of maturity, complexity, and the inability to standardize large-scale production. Could organoids therefore be a better choice for studying MetS than other models? How can these limitations be overcome? Here, we summarize the available data to present current progress on pancreatic and hepatobiliary organoids and to answer these open questions. Organoids are of human origin and contain a variety of human cell types necessary to mimic the disease characteristics of MetS in their development. Taken together with the discovery of hepatobiliary progenitors in situ, the dedifferentiation of beta cells in diabetes, and studies on hepatic macrophages, we suggest that promoting endogenous regeneration has the potential to prevent the development of end-stage liver and pancreatic lesions caused by MetS and outline the direction of future research in this field.

Hair Follicles as a Critical Model for Monitoring the Circadian Clock.

Clock (circadian) genes are heterogeneously expressed in hair follicles (HFs). The genes can be modulated by both the central circadian system and some extrinsic factors, such as light and thyroid hormones. These circadian genes participate in the regulation of several physiological processes of HFs, including hair growth and pigmentation. On the other hand, because peripheral circadian genes are synchronized with the central clock, HFs could provide a noninvasive and practical method for monitoring and evaluating multiple circadian-rhythm-related conditions and disorders among humans, including day and night shifts, sleep-wake disorders, physical activities, energy metabolism, and aging. However, due to the complexity of circadian biology, understanding how intrinsic oscillation operates using peripheral tissues only may be insufficient. Combining HF sampling with multidimensional assays such as detection of body temperature, blood samples, or certain validated questionnaires may be helpful in improving HF applications. Thus, HFs can serve as a critical model for monitoring the circadian clock and can help provide an understanding of the potential mechanisms of circadian-rhythm-related conditions; furthermore, chronotherapy could support personalized treatment scheduling based on the gene expression profile expressed in HFs.

High Expression of CD58 and ALDH1A3 Predicts a Poor Prognosis in Basal-like Breast Cancer.

BACKGROUND/AIM: CD58 is an immune adhesion molecule on the cellular surface. It was previously found that a high expression of CD58 predicted a poor prognosis of patients with lower-grade gliomas. Therefore, the aim of this paper was to investigate the association between CD58 and breast cancer. MATERIALS AND METHODS: CD58 gene expression data downloaded from cBioPortal was compared between the different subtypes of breast cancer. Clinical prognosis was examined using Kaplan-Meier analysis and multivariable Cox regression analysis. The association between CD58 expression and immune cell infiltration was estimated using the TIMER 2.0 web platform. Finally, the tumour sphere formation of aldehyde dehydrogenase 1 (ALDH1)high basal-like breast cancer stem cells in which CD58 was knocked down using siRNA was measured. RESULTS: CD58 mRNA was mainly enriched in claudin-low and basal-like subtypes. The high expression of CD58 predicted a good prognosis in patients with luminal A and luminal B breast cancer. This prediction may be due to the association of immune cell infiltration with CD58. Notably, patients with luminal A breast cancer with a high expression of CD58 in association with ALDH1A3 exhibited a good prognosis; however, this did not apply to patients with basal-like breast cancer. The in vitro experiments revealed that knockdown of CD58 inhibited the tumour sphere formation ability of ALDH1high basal-like cancer cells. CONCLUSION: CD58 may function as a potential prognostic biomarker and therapeutic target in ALDH-positive basal-like cancer stem cells.

Recognition of Melanocytes in Immuno-Neuroendocrinology and Circadian Rhythms: Beyond the Conventional Melanin Synthesis.

Melanocytes produce melanin to protect the skin from UV-B radiation. Notwithstanding, the spectrum of their functions extends far beyond their well-known role as melanin production factories. Melanocytes have been considered as sensory and computational cells. The neurotransmitters, neuropeptides, and other hormones produced by melanocytes make them part of the skin's well-orchestrated and complex neuroendocrine network, counteracting environmental stressors. Melanocytes can also actively mediate the epidermal immune response. Melanocytes are equipped with ectopic sensory systems similar to the eye and nose and can sense light and odor. The ubiquitous inner circadian rhythm controls the body's basic physiological processes. Light not only affects skin photoaging, but also regulates inner circadian rhythms and communicates with the local neuroendocrine system. Do melanocytes "see" light and play a unique role in photoentrainment of the local circadian clock system? Why, then, are melanocytes responsible for so many mysterious functions? Do these complex functional devices work to maintain homeostasis locally and throughout the body? In addition, melanocytes have also been shown to be localized in internal sites such as the inner ear, brain, and heart, locations not stimulated by sunlight. Thus, what can the observation of extracutaneous melanocytes tell us about the "secret identity" of melanocytes? While the answers to some of these intriguing questions remain to be discovered, here we summarize and weave a thread around available data to explore the established and potential roles of melanocytes in the biological communication of skin and systemic homeostasis, and elaborate on important open issues and propose ways forward.

Melanocyte stem cells in skin diseases and their potential in cell-based therapy.

Melanocytes have a complex function and play an important role in a variety of regulatory mechanisms in the human system. Melanocyte stem cells (MelSCs) serve as a reservoir to replenish the melanocytes by regenerating new ones, and they are capable of self-renewal and differentiation to maintain their homeostasis, repair, and regeneration in tissues. The numerical decrease and functional impairment of MelSCs may be closely related to the development and treatment response of many skin diseases. However, the current knowledge about MelSCs mainly comes from studies in mice, and little is known about human MelSC markers; especially, their markers are still unclear or lack consensus. This leads to uncertainty in clinical findings, which further limits our comprehensive understanding of pigmentary disorders and also hinders the progress of new treatments. Thus, in this review article, combined with our previous and current work, we summarize and update the recent advances in MelSC research, including the molecular markers of human MelSCs and their niche, as well as the association of MelSCs with skin diseases, including vitiligo, hair greying, and melanoma. Due to the limited tools available to explore the identified characteristics of human MelSCs, pluripotent stem cells can provide a new research model for further study, especially combined with CRISPR/Cas9 technology. The visualization of human MelSCs' development and differentiation can help to identify their molecular characteristics and understand their cellular fate dynamically, which will allow us not only to further explore their roles in associated diseases, but also to achieve MelSC-based cellular therapy.

Retinoids rescue ceruloplasmin secretion and alleviate oxidative stress in Wilson's disease-specific hepatocytes.

Wilson's disease (WD) is a copper metabolic disorder caused by a defective ATP7B function. Conventional therapies cause severe side effects and significant variation in efficacy, according to cohort studies. Thus, exploring new therapeutic approaches to prevent progression to liver failure is urgent. To study the physiology and pathology of WD, immortalized cell lines and rodent WD models have been used conventionally; however, a large gap remains among different species as well as in genetic backgrounds among individuals. We generated induced pluripotent stem cells (iPSCs) from four WD patients carrying compound heterozygous mutations in the ATP7B gene. ATP7B loss- and gain-of-functions were further manifested with ATP7B-deficient iPSCs and heterozygously corrected R778L WD patient-derived iPSCs using CRISPR-Cas9-based gene editing. Although the expression of ATP7B protein varied among WD-specific hepatocytes differentiated from these iPSCs, the expression and secretion of ceruloplasmin (Cp), a downstream copper carrier in plasma, were consistently decreased in WD patient-derived and ATP7B-deficient hepatocytes. A transcriptome analysis detected abnormalities in the retinoid signaling pathway and lipid metabolism in WD-specific hepatocytes. Drug screening using WD patient-derived hepatocytes identified retinoids as promising candidates for rescuing Cp secretion. All-trans retinoic acid also alleviates reactive oxygen species production induced by lipid accumulation in WD-specific hepatocytes treated with oleic acid. These patient-derived iPSC-based hepatic models function as effective platforms for the development of potential therapeutics for hepatic steatosis in WD and other fatty liver diseases.

Limitations and challenges of direct cell reprogramming in vitro and in vivo.

Direct reprogramming, whether in vitro or in vivo, has attracted great attention because of its advantages of convenience, short-term conversion, direct targets, no immune rejection, and potential clinical applications. In addition, due to its independence from the pluripotent state, direct programming minimizes some safety concerns associated with the use of human pluripotent stem cells. However, the significant limitations of reprogrammed cells, such as poor proliferative ability, low efficiency, and immature function, need to be addressed before the clinical application potential can be expanded. Here, we review the recent achievements of direct reprogramming in 2D and 3D systems in vitro and in vivo, covering cells derived from the three germ layers from stem/progenitor cells to terminal cells, such as hepatocytes, pancreatic ß cells, cardiomyocytes, endothelial cells, osteoblasts, chondrocytes, neurons, and melanocytes. Combining our lab experiences with current work, we summarize the practical and potential issues that need to be solved and the prospects of strategies for addressing the current dilemmas. Through comprehensive analyses, it is concluded that the directions for dealing with efficiency and functionality issues could be the optimization of transcription factors, the upgradation for delivery systems, the regulation of epigenetic factors and pathways, and the improvement of cellular maintenance conditions. Besides, converting cells into the progenitor state firstly and then differentiating them into the desired cell types with chemical compounds may provide an approach to obtaining functional and safe converted cells in batches with a better proliferative ability. With the emergence of more and more direct reprogramming techniques and approaches with both safety and effectiveness, it is bound to bring a new dawn for mechanism research and therapeutic applications for relevant diseases in the future.

Improving the repopulation capacity of elderly human hepatocytes by decoding aging-associated hepatocyte plasticity.

BACKGROUND AND AIMS: The loss of liver regenerative capacity is the most dramatic age-associated alteration. Because of an incomplete mechanistic understanding of the liver aging process, a successful therapeutic strategy to improve liver regeneration in the elderly has not been developed so far. Hepatocyte plasticity is a principal mechanism for producing new hepatocytes and cholangiocytes during regeneration. This study aims to promote the repopulation capacity of elderly hepatocytes by decoding the underlying mechanism about the regulation of aging on human hepatocyte plasticity. APPROACH AND RESULTS: To understand the age-related mechanisms, we established a hepatocyte aging model from human-induced pluripotent stem cells and developed a method for ex vivo characterization of hepatocyte plasticity. We found that hepatocyte plasticity was gradually diminished with aging, and the impaired plasticity was caused by age-induced histone hypoacetylation. Notably, selective inhibition of histone deacetylases could markedly restore aging-impaired plasticity. Based on these findings, we successfully improved the plasticity of elderly primary human hepatocytes that enhanced their repopulation capacity in the liver injury model. CONCLUSIONS: This study suggests that age-induced histone hypoacetylation impairs hepatocyte plasticity, and hepatocyte plasticity might be a therapeutic target for promoting the regenerative capacity of the elderly liver.

Gene Editing in Pluripotent Stem Cells and Their Derived Organoids.

With the rapid rise in gene-editing technology, pluripotent stem cells (PSCs) and their derived organoids have increasingly broader and practical applications in regenerative medicine. Gene-editing technologies, from large-scale nucleic acid endonucleases to CRISPR, have ignited a global research and development boom with significant implications in regenerative medicine. The development of regenerative medicine technologies, regardless of whether it is PSCs or gene editing, is consistently met with controversy. Are the tools for rewriting the code of life a boon to humanity or a Pandora's box? These technologies raise concerns regarding ethical issues, unexpected mutations, viral infection, etc. These concerns remain even as new treatments emerge. However, the potential negatives cannot obscure the virtues of PSC gene editing, which have, and will continue to, benefit mankind at an unprecedented rate. Here, we briefly introduce current gene-editing technology and its application in PSCs and their derived organoids, while addressing ethical concerns and safety risks and discussing the latest progress in PSC gene editing. Gene editing in PSCs creates visualized in vitro models, providing opportunities for examining mechanisms of known and unknown mutations and offering new possibilities for the treatment of cancer, genetic diseases, and other serious or refractory disorders. From model construction to treatment exploration, the important role of PSCs combined with gene editing in basic and clinical medicine studies is illustrated. The applications, characteristics, and existing challenges are summarized in combination with our lab experiences in this field in an effort to help gene-editing technology better serve humans in a regulated manner. Current preclinical and clinical trials have demonstrated initial safety and efficacy of PSC gene editing; however, for better application in clinical settings, additional investigation is warranted.

Generation of human induced pluripotent stem cell lines carrying homozygous JAG1 deletions.

JAG1gene encodes Jagged1 protein, which is a ligand for NOTCH receptors. JAG1 mutations cause Alagille syndrome, in which liver failure occurs caused by abnormalities in the bile ducts. In this study, we generated two homozygous JAG1 knockout iPSC lines (JAG1KO iPSC) by creating indels with CRISPR-Cas9 technology. These newly generated JAG1KO iPSC lines showed similar self-renewal and pluripotency as their original iPSC WTC11 line. These iPSC lines carried deletions around the translation start codon of JAG1 gene, causing compromised Jagged1 protein expression. These JAG1KO iPSC lines are promising bioresources to studyJagged1 function in human development and pathology.

Current trends in three-dimensional visualization and real-time navigation as well as robot-assisted technologies in hepatobiliary surgery.

With the continuous development of digital medicine, minimally invasive precision and safety have become the primary development trends in hepatobiliary surgery. Due to the specificity and complexity of hepatobiliary surgery, traditional preoperative imaging techniques such as computed tomography and magnetic resonance imaging cannot meet the need for identification of fine anatomical regions. Imaging-based three-dimensional (3D) reconstruction, virtual simulation of surgery and 3D printing optimize the surgical plan through preoperative assessment, improving the controllability and safety of intraoperative operations, and in difficult-to-reach areas of the posterior and superior liver, assistive robots reproduce the surgeon's natural movements with stable cameras, reducing natural vibrations. Electromagnetic navigation in abdominal surgery solves the problem of conventional surgery still relying on direct visual observation or preoperative image assessment. We summarize and compare these recent trends in digital medical solutions for the future development and refinement of digital medicine in hepatobiliary surgery.

The Effect of the Platelet Administration for the Patients with Liver Dysfunction after Liver Resection: Preliminary Clinical Trial.

The aim of this study is to investigate the effect of platelet on the improvement of deteriorated liver function after liver resection. Six patients with hepatocellular carcinoma and liver cirrhosis have received the partial hepatectomy in the institution. Their Child-Pugh grade was B, and platelet count was below 7,000/µl. After hepatectomy, 20 units of platelet transfusion were carried out, liver function and side effects were investigated after 4 weeks, and the number of platelets increased to approximately 15,000/µl. Liver functions, such as aspartate transaminase (AST), alanine aminotransferase (ALT), cholinesterase (ChE), and prothrombin time, as well as albumin, recover to the same level as those before operation and 4 weeks after the operation. Any side effects were not recognized in all patients. Administration of platelets for cirrhotic patient with hepatectomy was carried with safety. But remarkable effect on the improvement of liver function was not recognized.

Survival-Assured Liver Injury Preconditioning (SALIC) Enables Robust Expansion of Human Hepatocytes in Fah-/- Rag2-/- IL2rg-/- Rats.

Although liver-humanized animals are desirable tools for drug development and expansion of human hepatocytes in large quantities, their development is restricted to mice. In animals larger than mice, a precondition for efficient liver humanization remains preliminary because of different xeno-repopulation kinetics in livers of larger sizes. Since rats are ten times larger than mice and widely used in pharmacological studies, liver-humanized rats are more preferable. Here, Fah-/- Rag2-/- IL2rg-/- (FRG) rats are generated by CRISPR/Cas9, showing accelerated liver failure and lagged liver xeno-repopulation compared to FRG mice. A survival-assured liver injury preconditioning (SALIC) protocol, which consists of retrorsine pretreatment and cycling 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) administration by defined concentrations and time intervals, is developed to reduce the mortality of FRG rats and induce a regenerative microenvironment for xeno-repopulation. Human hepatocyte repopulation is boosted to 31 ± 4% in rat livers at 7 months after transplantation, equivalent to approximately a 1200-fold expansion. Human liver features of transcriptome and zonation are reproduced in humanized rats. Remarkably, they provide sufficient samples for the pharmacokinetic profiling of human-specific metabolites. This model is thus preferred for pharmacological studies and human hepatocyte production. SALIC may also be informative to hepatocyte transplantation in other large-sized species.

Hepatitis B virus infection modeling using multi-cellular organoids derived from human induced pluripotent stem cells.

Chronic infection with hepatitis B virus (HBV) remains a global health concern despite the availability of vaccines. To date, the development of effective treatments has been severely hampered by the lack of reliable, reproducible, and scalable in vitro modeling systems that precisely recapitulate the virus life cycle and represent virus-host interactions. With the progressive understanding of liver organogenesis mechanisms, the development of human induced pluripotent stem cell (iPSC)-derived hepatic sources and stromal cellular compositions provides novel strategies for personalized modeling and treatment of liver disease. Further, advancements in three-dimensional culture of self-organized liver-like organoids considerably promote in vitro modeling of intact human liver tissue, in terms of both hepatic function and other physiological characteristics. Combined with our experiences in the investigation of HBV infections using liver organoids, we have summarized the advances in modeling reported thus far and discussed the limitations and ongoing challenges in the application of liver organoids, particularly those with multi-cellular components derived from human iPSCs. This review provides general guidelines for establishing clinical-grade iPSC-derived multi-cellular organoids in modeling personalized hepatitis virus infection and other liver diseases, as well as drug testing and transplantation therapy.

The evidence of a macrophage barrier in the xenotransplantation of human hematopoietic stem cells to severely immunodeficient rats.

BACKGROUND: The human-to-rat hematopoietic stem cell transplantation (HSCT) model is rare, unlike its human-to-mouse counterpart. The rat models are desired, especially in areas of physiology, toxicology, and pharmacology. In addition to lymphocytes, macrophages are also considered to be important for xenotransplantation. We generated a rat xenotransplantation model to prove the role of macrophages as a xenotransplantation barrier. METHODS: Immunodeficiency in SRG rats, which are Sprague-Dawley (SD) rats lacking Rag2 and Il2rg, was confirmed by flow cytometry and spleen immunostaining. Human umbilical cord blood was collected after scheduled cesarean section at the University of Tsukuba Hospital. Cord blood mononuclear cells (CB-MNCs) were transplanted into the SRG rats administered several injections of clodronate liposome (CL), which cause macrophage depletion. Survival of human cells was observed by flow cytometry. Rat macrophage phagocytosis assay was performed to check the species-specific effects of rat macrophages on injected human/rat blood cells. RESULTS: SRG rats were deficient in T/B/NK cells. Without CL pretreatment, human CB-MNCs were removed from SRG rats within 7 hours after transplantation. The rats pretreated with CL could survive after transplantation. Prolonged survival for more than 4 weeks was observed only following a one-time CL injection. Rat macrophages had a species-specific potential for the phagocytosis of human blood cells in vivo. CONCLUSION: In human-to-rat HSCT, the short period of early macrophage control, leading to macrophage immunotolerance, is important for engraftment. The generated model can be useful for the creation of future xenotransplantation models or other clinical research.