Technical considerations and strategies for generating and optimizing humanized mouse tumor models in immuno-oncology research.

The field of cancer immunotherapy has experienced significant progress, resulting in the emergence of numerous biological drug candidates requiring in vivo efficacy testing and a better understanding of their mechanism of action (MOA). Humanized immune system (HIS) models are valuable tools in this regard. However, there is a lack of systematic guidance on HIS modeling. To address this issue, the present study aimed to establish and optimize a variety of HIS models for immune-oncology (IO) study, including genetically engineered mouse models and HIS models with human immune components reconstituted in severely immunocompromised mice. The efficacy and utility of these models were tested with several marketed or investigational IO drugs according to their MOA, followed by immunophenotypic analysis and efficacy evaluation. The results of the present study demonstrated that the HIS models responded to various IO drugs as expected and that each model had unique niches, utilities and limitations. Researchers should carefully choose the appropriate models based on the MOA and the targeted immune cell populations of the investigational drug. The present study provides valuable methodologies and actionable technical guidance on designing, generating or utilizing appropriate HIS models to address specific questions in translational IO.

HAT1 signaling confers to assembly and epigenetic regulation of HBV cccDNA minichromosome.

Rationale: Hepatitis B virus (HBV) is a leading cause of liver diseases. HBV covalently closed circular DNA (cccDNA) is a critical obstacle of complete elimination by anti-HBV therapy. HBV cccDNA accumulates in nucleus as a chromatin-like cccDNA minichromosome assembled by histones and non-histones. However, the underlying mechanism of modulation of cccDNA minichromosome in hepatocytes is poorly understood. Methods: A human liver-chimeric mouse model was established. The cccDNA-ChIP, Southern blot analysis, confocal assays, RIP assays and RNA pull-down assays, et al. were performed to assess the mechanism of assembly and epigenetic regulation of cccDNA minichromosome in human liver-chimeric mouse model, human primary hepatocytes (PHH), dHepaRG, HepG2-NTCP cell lines and clinical liver tissues. Results: Importantly, the expression levels of HAT1, CAF-1 and lncRNA HULC were significantly elevated in the liver from HBV-infected human liver-chimeric mice. Strikingly, the depletion of HAT1 reduced HBV replication and cccDNA accumulation, and impaired the assembly of histone H3/H4 and the deposition of HBx and p300 onto cccDNA to form cccDNA minichromosome in the cells. Mechanically, chromatin assembly factor-1 (CAF-1) was involved in the events. Interestingly, HAT1 modified the acetylation of histone H3K27/H4K5/H4K12 on cccDNA minichromosome. Moreover, lncRNA HULC-scaffold HAT1/HULC/HBc complex was responsible for the modification on cccDNA minichromosome. Additionally, HBV activated HAT1 through HBx-co-activated transcriptional factor Sp1 in a positive feedback manner. Conclusion: HAT1 signaling contributes to assembly and epigenetic regulation of HBV cccDNA minichromosome.

LncRNA PCNAP1 modulates hepatitis B virus replication and enhances tumor growth of liver cancer.

Rationale: Hepatitis B virus (HBV) is a major risk factor for liver cancer, in which HBV covalently closed circular DNA (cccDNA) plays crucial roles. However, the effect of pseudogene-derived long noncoding RNAs (lncRNAs) acting as functional regulators of their ancestral gene expression on HBV replication and hepatocellular carcinoma (HCC) remains unclear. In this study, we speculated that the pseudogene-derived lncRNA PCNAP1 and its ancestor PCNA might modulate HBV replication and promote hepatocarcinogenesis. Methods: We investigated the roles of lncRNA PCNAP1 in contribution of HBV replication through modulating miR-154/PCNA/HBV cccDNA signaling in hepatocarcinogenesis by using CRISPR/Cas9, Southern blot analysis, confocal assays, et al. in primary human hepatocytes (PHH), HepaRG cells, HepG2-NTCP cells, hepatoma carcinoma cells, human liver-chimeric mice model, transgenetic mice model, in vitro tumorigenicity and clinical patients. Results: Interestingly, the expression levels of PCNAP1 and PCNA were significantly elevated in the liver of HBV-infectious human liver-chimeric mice. Clinically, the mRNA levels of PCNAP1 and PCNA were increased in the liver of HBV-positive/HBV cccDNA-positive HCC patients. Mechanistically, PCNA interacted with HBV cccDNA in a HBc-dependent manner. PCNAP1 enhanced PCNA through sponging miR-154 targeting PCNA mRNA 3'UTR. Functionally, PCNAP1 or PCNA remarkably enhanced HBV replication and accelerated the growth of HCC in vitro and in vivo. Conclusion: We conclude that lncRNA PCNAP1 enhances the HBV replication through modulating miR-154/PCNA/HBV cccDNA signaling and the PCNAP1/PCNA signaling drives the hepatocarcinogenesis. Our finding provides new insights into the mechanism by which lncRNA PCNAP1 enhances HBV replication and hepatocarcinogenesis.

miR-22 Inhibits CD34+ Cell Expansion Through Decreasing ß-Catenin in Osteoblasts.

The bone marrow (BM) microenvironment, heavily composed of osteoblasts, plays a key role during the normal development of hematopoiesis. Endogenous miR-22 has an important function in the hematopoietic development and osteoblastic differentiation. It is unclear whether miR-22 in osteoblasts from the BM microenvironment also has an important function in the development of hematopoiesis. This study found that the capacity of hTERT-transduced fetal bone marrow osteoblasts (FBMOB-hTERT) cells to expand human cord blood (CB) CD34+ cells and maintain the multipotency of CB CD34+ cells is decreased upon ectopic expression of miR-22. Further experiments revealed that with the existence of CB CD34+ cells, the expression of ß-catenin in FBMOB-hTERT cells is decreased upon ectopic expression of miR-22. The reduced ability of FBMOB-hTERT cells to expand human CB CD34+ cells and maintain the multipotency of CB CD34+ cells upon ectopic miR-22 was partly rescued by overexpression of ß-catenin. The study indicated that the ability of osteoblasts to expand human CB CD34+ cells and maintain the multipotency of CB CD34+ cells is decreased upon ectopic expression of miR-22. The decreased expression of ß-catenin is, at least partly, responsible for the reduced ability of osteoblasts for expanding and supporting CB CD34+ cells upon ectopic expression of miR-22.

miR-17 promotes expansion and adhesion of human cord blood CD34(+) cells in vitro.

INTRODUCTION: We have recently found that miR-17 is necessary in the cell-extrinsic control of cord blood (CB) CD34(+) cell function. Here, we demonstrated that the proper level of miR-17 is also necessary in the cell-intrinsic control of the hematopoietic properties of CB CD34(+) cells. METHODS: The miR-17 overexpression and knockdown models were created using primary CB CD34(+) cells transfected by the indicated vectors. Long-term culture, colony forming, adhesion and trans-well migration assays were carried out to investigate the function of miR-17 on CB CD34(+) cells in vitro. NOD prkdc (scid) Il2rg (null) mice were used in a SCID repopulating cell assay to investigate the function of miR-17 on CB CD34(+) cells in vivo. A two-tailed Student's t-test was used for statistical comparisons. RESULTS: In vitro assays revealed that ectopic expression of miR-17 promoted long-term expansion, especially in the colony-forming of CB CD34(+) cells and CD34(+)CD38(-) cells. Conversely, downregulation of miR-17 inhibited the expansion of CB CD34(+) cells. However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells. The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice. CONCLUSION: We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

Generation of naive induced pluripotent stem cells from rhesus monkey fibroblasts.

Conventional embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) derived from primates resemble mouse epiblast stem cells, raising an intriguing question regarding whether the naive pluripotent state resembling mouse embryonic stem cells (mESCs) exists in primates and how to capture it in vitro. Here we identified several specific signaling modulators that are sufficient to generate rhesus monkey fibroblast-derived iPSCs with the features of naive pluripotency in terms of growth properties, gene expression profiles, self-renewal signaling, X-reactivation, and the potential to generate cross-species chimeric embryos. Interestingly, together with recent reports of naive human pluripotent stem cells, our findings suggest several conserved signaling pathways shared with rodents and specific to primates, providing significant insights for acquiring naive pluripotency from other species. In addition, the derivation of rhesus monkey naive iPSCs also provides a valuable cell source for use in preclinical research and disease modeling.

Directed differentiation of human embryonic stem cells into thymic epithelial progenitor-like cells reconstitutes the thymic microenvironment in vivo.

Thymus transplantation has great clinical potential for treating immunological disorders, but the shortage of transplant donors limits the progress of this therapy. Human embryonic stem cells (hESCs) are promising cell sources for generating thymic epithelial cells. Here, we report a stepwise protocol to direct the differentiation of hESCs into thymic epithelial progenitor-like cells (TEPLCs) by mimicking thymus organogenesis with sequential regulation of Activin, retinoic acid, BMP, and WNT signals. The hESC-derived TEPLCs expressed the key thymic marker gene FOXN1 and could further develop in vivo into thymic epithelium expressing the functional thymic markers MHC II and AIRE upon transplantation. Moreover, the TEPLC-derived thymic epithelium could support mouse thymopoiesis in T-cell-deficient mice and promote human T cell generation in NOD/SCID mice engrafted with human hematopoietic stem cells (hHSCs). These findings could facilitate hESC-based replacement therapy and provide a valuable in vitro platform for studying human thymus organogenesis and regeneration.

TGFß inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy.

Embryonic hematopoiesis is a complex process. Elucidating the mechanism regulating hematopoietic differentiation from pluripotent stem cells would allow us to establish a strategy to efficiently generate hematopoietic cells. However, the mechanism governing the generation of hematopoietic progenitors from human embryonic stem cells (hESCs) remains unknown. Here, on the basis of the emergence of CD43(+) hematopoietic cells from hemogenic endothelial (HE) cells, we demonstrated that VEGF was essential and sufficient, and that bFGF was synergistic with VEGF to specify the HE cells and the subsequent transition into CD43(+) hematopoietic cells. Significantly, we identified TGFß as a novel signal to regulate hematopoietic development, as the TGFß inhibitor SB 431542 significantly promoted the transition from HE cells into CD43(+) hematopoietic progenitor cells (HPCs) during hESC differentiation. By defining these critical signaling factors during hematopoietic differentiation, we can efficiently generate HPCs from hESCs. Our strategy could offer an in vitro model to study early human hematopoietic development.

Retinoic acid enhances the generation of hematopoietic progenitors from human embryonic stem cell-derived hemato-vascular precursors.

Current induction schemes directing hematopoietic differentiation of human embryonic stem cells (hESCs) are not well defined to mimic the sequential stages of hematopoietic development in vivo. Here, we report a 3-stage method to direct differentiation of hESCs toward hematopoietic progenitors in chemically defined mediums. In the first 2 stages, we efficiently generated T-positive primitive streak/mesendoderm cells and kinase domain receptor-positive (KDR(+)) platelet-derived growth factor receptor α-negative (PDGFRα(-)) hemato-vascular precursors sequentially. In the third stage, we found that cells in a spontaneous differentiation condition mainly formed erythroid colonies. Addition of all-trans retinoic acid (RA) greatly enhanced generation of hematopoietic progenitors in this stage while suppressing erythroid development. The RA-treated cells highly expressed definitive hematopoietic genes, formed large numbers of multilineage and myeloid colonies, and gave rise to greater than 45% CD45(+) hematopoietic cells. When hematopoietic progenitors were selected with CD34 and C-Kit, greater than 95% CD45(+) hematopoietic cells could be generated. In addition, we found that endogenous RA signaling at the second stage was required for vascular endothelial growth factor/basic fibroblast growth factor-induced hemato-vascular specification, whereas exogenously applied RA efficiently induced KDR(-)PDGFRα(+) paraxial mesoderm cells. Our study suggests that RA signaling plays diverse roles in human mesoderm and hematopoietic development.

Establishment and characterization of a human telomerase catalytic subunit-transduced fetal bone marrow-derived osteoblastic cell line.

The fate of human hematopoietic stem cells (HSCs)/progenitor cells (HPCs) is influenced by bone marrow (BM) stromal cells. To investigate the role of stromal cells in the hematopoietic support, we have transduced human fetal BM stromal cells (FBMSCs) with a human telomerase catalytic subunit (hTERT). One of the resultant cell lines was identified as osteoblasts, because it contained mineral deposits and constitutively expressed osteogenic genes osteocalcin, osteopontin, collagen type I, osteoblast marker alkaline phosphatase, but not marrow stromal cell marker STRO-1 and CD105. The hTERT-transduced fetal BM-derived osteoblastic cells (FBMOB-hTERT) can actively maintain the capacity of self-renewal and multipotency of HSCs/HPCs at least partly through transcriptional up-regulation of hematopoietic growth factors such as stem cell growth factors (SCFs) and Wnt-5A during interaction with HSCs/HPCs. The enhanced transcription of SCFs and Wnt-5A appears to be mediated by CD29 signaling. Moreover, the FBMOB-hTERT cells seem superior to primary FBMSCs in supporting hematopoiesis, because they are more potent than primary FBMSCs in supporting the ex vivo expansion and long-term culture initiating cells activity of HSCs. The FBMOB-hTERT cell line has been maintained in vitro more than 125 population doublings without tumorigenicity. The results indicate that the FBMOB-hTERT is useful for the study of molecular mechanisms by which osteoblasts support hematopoiesis.

The existence of epithelial-to-mesenchymal cells with the ability to support hematopoiesis in human fetal liver.

The fetal liver is the major hematopoietic organ during mid-gestation, and it is also a source of stem cells that exist in a complex environment. In this study, we isolated a population of actively replicating cells with the characteristic of the epithelial-to-mesenchymal transition (EMT) from fetal liver. These cells were identified with the epithelial markers, including alpha-fetoprotein (AFP), albumin (ALB), cytokeratins (CK) 7, and CK18, as well as the mesenchymal markers, such as alpha-smooth muscle actin (ASMA), CD29, CD44, CD49, CD54, collagen I and osteopontin (OPN). Furthermore, they also expressed some hematopoiesis-related genes. In addition, the cell population had the ability to retain hematopoietic stem cells (HSCs) in an undifferentiated state in vitro during cytokine-stimulated proliferation. These results provide an insight about early human liver development and may also help to understand hematopoiesis in the fetal liver.

Chitosan/gelatin composite microcarrier for hepatocyte culture.

Solid and porous chitosan/gelatin (CG) composite microcarriers were prepared by a water-in-oil emulsion process with additional freezing and lyophilization. Adult rat hepatocytes (10(6) cells ml(-1)) attached on CG microcarriers maintained at least 15 d of viability and differentiated functions. Over 15 d, unimmobilized hepatocytes released 1.34-fold less lactate dehydrogenase (LDH), and retained 1.63-, 1.51- and 1.28-fold higher albumin secretion, urea synthesis and 7-ethoxycoumarin deethylation activities, respectively, than those on collagen-coated microcarriers. The CG matrix is therefore a promising microcarrier for hepatocyte culture.

[Major role of stromal cell derived factor and its receptor CXCR4 in the mobilization and homing of hematopoietic stem/progenitor cells - review].

Stromal cell derived factor (SDF), expressing on bone marrow stromal cells is a CXC-type chemokine, which specifically chemoattracts hematopoietic stem cells (HSCs) expressing CXCR4. SDF plays important roles in homing and mobilizing of HSCs. In this paper the regulatory mechanism of SDF/CXCR4 in the HSC migration process is mainly reviewed.