Hox-driven conditional immortalization of myeloid and lymphoid progenitors: Uses, advantages, and future potential.

Those who study macrophage biology struggle with the decision whether to utilize primary macrophages derived directly from mice or opt for the convenience and genetic tractability of immortalized macrophage-like cell lines in in vitro studies. Particularly when it comes to studying phagocytosis and phagosomal maturation-a signature cellular process of the macrophage-many commonly used cell lines are not representative of what occurs in primary macrophages. A system developed by Mark Kamps' group, that utilizes conditionally constitutive activity of Hox transcription factors (Hoxb8 and Hoxa9) to immortalize differentiation-competent myeloid cell progenitors of mice, offers an alternative to the macrophage/macrophage-like dichotomy. In this resource, we will review the use of Hoxb8 and Hoxa9 as hematopoietic regulators to conditionally immortalize murine hematopoietic progenitor cells which retain their ability to differentiate into many functional immune cell types including macrophages, neutrophils, basophils, osteoclasts, eosinophils, dendritic cells, as well as limited potential for the generation of lymphocytes. We further demonstrate that the use of macrophages derived from Hoxb8/Hoxa9 immortalized progenitors and their similarities to bone marrow-derived macrophages. To supplement the existing data, mass spectrometry-based proteomics, flow cytometry, cytology, and in vitro phagosomal assays were conducted on macrophages derived from Hoxb8 immortalized progenitors and compared to bone marrow-derived macrophages and the macrophage-like cell line J774. We additionally propose the use of a standardized nomenclature to describe cells derived from the Hoxb8/Hoxa9 system in anticipation of their expanded use in the study of leukocyte cell biology.

Establishment of a conditionally immortalized mouse optic nerve astrocyte line.

Optic nerve astrocytes play a major role in axonal degeneration and regeneration. Astrocyte lines are an important tool to elucidate the responsible cellular mechanisms. In this study, we established a conditionally immortalized mouse optic nerve astrocyte line. Astrocytes were cultured from explants derived from postnatal day 4-5 H-2kb-tsA58 transgenic mouse optic nerves. Cells were cultured in defined astrocyte culture medium under permissive (33 °C) or non-permissive (38.5 °C) temperatures with or without interferon-ɤ (IFN-ɤ). Astrocytes were characterized by immunocytochemistry staining using antibodies against glial fibrillary acidic protein (GFAP) and neural cell adhesion molecule (NCAM). Cell proliferation rates were determined by cell growth curves and percentage of Ki67 positive cells. Karyotyping was performed to validate the mouse origin of established cell line. Conditional immortalization was assessed by western blot-determined expression levels of SV40 large T antigen (TAg), p53, GFAP and NCAM in non-permissive culture conditions. In addition, phagocytic activity of immortalized cells was determined by flow cytometry-based pHrodo fluorescence analysis. After 5 days in culture, cells migrated out from optic nerve explants. Immunocytochemistry staining showed that migrating cells expressed astrocyte makers, GFAP and NCAM. In permissive conditions, astrocytes had increased expression levels of TAg and p53, exhibited a greater cell proliferation rate as well as a higher percentage of Ki67 positive cells (n = 3, p < 0.05) compared to cells cultured in non-permissive conditions. One cell line (ImB1ON) was further maintained through 60 generations. Karyotyping showed that ImB1ON was of mouse origin. Flow cytometry-based pHrodo fluorescence analysis demonstrated phagocytic activity of ImB1ON cells. Quantitative PCR showed mRNA expression of trophic factors. Non-permissive culture conditions decreased expression of TAg and p53 in ImB1ON, and increased the expression of NCAM. A conditionally immortalized mouse optic nerve astrocyte line was established. This cell line provides an important tool to study astrocyte biological processes.

Conditionally immortalized human pancreatic stellate cell lines demonstrate enhanced proliferation and migration in response to IGF-I.

Pancreatic stellate cells (PSCs) play a key role in the dense desmoplastic stroma associated with pancreatic ductal adenocarcinoma. Studies on human PSCs have been minimal due to difficulty in maintaining primary PSC in culture. We have generated the first conditionally immortalized human non-tumor (NPSC) and tumor-derived (TPSC) pancreatic stellate cells via transformation with the temperature-sensitive SV40 large T antigen and human telomerase (hTERT). These cells proliferate at 33°C. After transfer to 37°C, the SV40LT is switched off and the cells regain their primary PSC phenotype and growth characteristics. NPSC contained cytoplasmic vitamin A-storing lipid droplets, while both NPSC and TPSC expressed the characteristic markers αSMA, vimentin, desmin and GFAP. Proteome array analysis revealed that of the 55 evaluated proteins, 27 (49%) were upregulated ≥3-fold in TPSC compared to NPSC, including uPA, pentraxin-3, endoglin and endothelin-1. Two insulin-like growth factor binding proteins (IGFBPs) were inversely expressed. Although discordant IGFBP-2 and IGFBP-3 levels, IGF-I was found to stimulate proliferation of both NPSC and TPSC. Both basal and IGF-I stimulated motility was significantly enhanced in TPSC compared to NPSC. In conclusion, these cells provide a unique resource that will facilitate further study of the active stroma compartment associated with pancreatic cancer.

The Derivation and Use of HoxB8-Driven Conditionally Immortalized Macrophages.

The use of Hox-driven conditionally immortalized immune cells has significantly increased in biomedical research over the past 15 years. HoxB8-driven conditionally immortalized myeloid progenitor cells maintain their ability to differentiate into functional macrophages. There are multiple benefits to this conditional immortalization strategy including the ability for unlimited propagation, genetic mutability, primary-like immune cells (macrophages, dendritic cells, and granulocytes) on demand, derivation from variety of mouse strains, and simple cryopreservation and reconstitution. In this chapter, we will discuss how to derive and use these HoxB8-conditionally immortalized myeloid progenitor cells.

In Vitro Generation of Murine Dendritic Cells from Hoxb8-Immortalized Hematopoietic Progenitors.

Mouse dendritic cells (DCs) are routinely generated based on cells isolated form the bone marrow (BM) and cultured in the presence of growth factors that support DC development, such as FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Guo et al., J Immunol Methods 432:24-29, 2016). In response to these growth factors, DC progenitors expand and differentiate, while other cell types die during the in vitro culture period, ultimately leading to relatively homogenous DC populations. An alternative method, which is discussed in detail in this chapter, relies on conditional immortalization of progenitor cells with DC potential in vitro using an estrogen-regulated form of Hoxb8 (ERHBD-Hoxb8). Such progenitors are established by retroviral transduction of largely unseparated BM cells with a retroviral vector expressing ERHBD-Hoxb8. Treatment of ERHBD-Hoxb8-expressing progenitors with estrogen results in Hoxb8 activation, which blocks cell differentiation and allows for expansion of homogenous progenitor cell populations in the presence of FLT3L. These cells, referred to as Hoxb8-FL cells, retain lineage potential for lymphocyte and myeloid lineages, including the DC lineage. Upon removal of estrogen (inactivation of Hoxb8), Hoxb8-FL cells differentiate into highly homogenous DC populations in the presence of GM-CSF or FLT3L akin to their endogenous counterparts. Given their unlimited proliferative capacity and amenability for genetic manipulation, for example, by CRISPR/Cas9, these cells provide a large number of options to investigate DC biology. Here, I am describing the method to establish Hoxb8-FL cells from mouse BM, as well as procedures for DC generation and gene deletion using lentivirally delivered CRISPR/Cas9.

Modeling colorectal tumorigenesis using the organoids derived from conditionally immortalized mouse intestinal crypt cells (ciMICs).

Intestinal cancers are developed from intestinal epithelial stem cells (ISCs) in intestinal crypts through a multi-step process involved in genetic mutations of oncogenes and tumor suppressor genes. ISCs play a key role in maintaining the homeostasis of gut epithelium. In 2009, Sato et al established a three-dimensional culture system, which mimicked the niche microenvironment by employing the niche factors, and successfully grew crypt ISCs into organoids or Mini-guts in vitro. Since then, the intestinal organoid technology has been used to delineate cellular signaling in ISC biology. However, the cultured organoids consist of heterogeneous cell populations, and it was technically challenging to introduce genomic changes into three-dimensional organoids. Thus, there was a technical necessity to develop a two-dimensional ISC culture system for effective genomic manipulations. In this study, we established a conditionally immortalized mouse intestinal crypt (ciMIC) cell line by using a piggyBac transposon-based SV40 T antigen expression system. We showed that the ciMICs maintained long-term proliferative activity under two-dimensional niche factor-containing culture condition, retained the biological characteristics of intestinal epithelial stem cells, and could form intestinal organoids in three-dimensional culture. While in vivo cell implantation tests indicated that the ciMICs were non-tumorigenic, the ciMICs overexpressing oncogenic ß-catenin and/or KRAS exhibited high proliferative activity and developed intestinal adenoma-like pathological features in vivo. Collectively, these findings strongly suggested that the engineered ciMICs should be used as a valuable tool cell line to dissect the genetic and/or epigenetic underpinnings of intestinal tumorigenesis.

Generation, Characterization, and Application of Inducible Proliferative Adult Human Epicardium-Derived Cells.

RATIONALE: In recent decades, the great potential of human epicardium-derived cells (EPDCs) as an endogenous cell source for cardiac regeneration has been recognized. The limited availability and low proliferation capacity of primary human EPDCs and phenotypic differences between EPDCs obtained from different individuals hampers their reproducible use for experimental studies. AIM: To generate and characterize inducible proliferative adult human EPDCs for use in fundamental and applied research. METHODS AND RESULTS: Inducible proliferation of human EPDCs was achieved by doxycycline-controlled expression of simian virus 40 large T antigen (LT) with a repressor-based lentiviral Tet-On system. In the presence of doxycycline, these inducible EPDCs (iEPDCs) displayed high and long-term proliferation capacity. After doxycycline removal, LT expression ceased and the iEPDCs regained their cuboidal epithelial morphology. Similar to primary EPDCs, iEPDCs underwent an epithelial-to-mesenchymal transition (EMT) after stimulation with transforming growth factor ß3. This was confirmed by reverse transcription-quantitative polymerase chain reaction analysis of epithelial and mesenchymal marker gene expression and (immuno) cytochemical staining. Collagen gel-based cell invasion assays demonstrated that mesenchymal iEPDCs, like primary EPDCs, possess increased invasion and migration capacities as compared to their epithelial counterparts. Mesenchymal iEPDCs co-cultured with sympathetic ganglia stimulated neurite outgrowth similarly to primary EPDCs. CONCLUSION: Using an inducible LT expression system, inducible proliferative adult human EPDCs were generated displaying high proliferative capacity in the presence of doxycycline. These iEPDCs maintain essential epicardial characteristics with respect to morphology, EMT ability, and paracrine signaling following doxycycline removal. This renders iEPDCs a highly useful new in vitro model for studying human epicardial properties.

Production and Characterization of a Conditionally Immortalized Dog Beta-Cell Line from Fetal Canine Pancreas.

Since the 1970s, rodent and human insulin-secreting pancreatic beta-cell lines have been developed and found useful for studying beta-cell biology. Surprisingly, although the dog has been widely used as a translational model for diabetes, no canine insulin-secreting beta cells have ever been produced. Here, a targeted oncogenesis protocol previously described by some of us for generating human beta cells was adapted to produce canine beta cells. Canine fetal pancreata were obtained by cesarean section between 42 and 55 days of gestation, and fragments of fetal glands were transduced with a lentiviral vector expressing SV40LT under the control of the insulin promoter. Two Lox P sites flanking the sequence allowed subsequent transgene excision by Cre recombinase expression. When grafted into SCID mice, these transduced pancreata formed insulinomas. ACT-164 is the cell line described in this report. Insulin mRNA expression and protein content were lower than reported with adult cells, but the ACT-164 cells were functional, and their insulin production in vitro increased under glucose stimulation. Transgene excision upon Cre expression arrested proliferation and enhanced insulin expression and production. When grafted in SCID mice, intact and excised cells reversed chemically induced diabetes. We have thus produced an excisable canine beta-cell line. These cells may play an important role in the study of several aspects of the cell transplantation procedure including the encapsulation process, which is difficult to investigate in rodents. Although much more work is needed to improve the excision procedure and achieve 100% removal of large T antigen expression, we have shown that functional cells can be obtained and might in the future be used for replacement therapy in diabetic dogs.

Conditional Immortalization of Lymphoid Progenitors via Tetracycline-Regulated LMO2 Expression.

Conditional immortalization of hematopoietic progenitors through lentiviral expression of selected transcription factors in hematopoietic stem and progenitor cells provides a promising tool to study stem cell and leukemia biology. In this study, to generate conditionally immortalized lymphoid progenitor (ciLP) cell lines, murine hematopoietic progenitor cells were transduced with an inducible lentiviral "all-in-one" vector expressing LMO2 under doxycycline (DOX) stimulation and the reverse tetracycline-regulated transactivator (rtTA3). For selection of LMO2-expressing ciLPs (LMO2-ciLPs) and longitudinal manipulation in T cell differentiation lymphoid conditions, we developed a robust approach based on coculture with OP9-DL1 stromal cells and improved cytokine conditions allowing a controlled balance between cell proliferation and differentiation in vitro. LMO2-ciLP cell lines with the highest proliferation, vector copy number, and similar insertion pattern were selected for LMO2 "on/off" in vitro study. LMO2 expression under DOX induction resulted in a double negative (DN) 2 differentiation arrest and a propagation of CD44+CD25- myeloid cell population characterized by lymphoid and myeloid phenotypes, respectively. Both DN2 and CD44+CD25- myeloid cell subpopulations expressed c-KIT, suggesting that LMO2-ciLPs were similar to uncommitted progenitors under DOX supplementation. DOX removal resulted in cessation of ectopic LMO2 expression and LMO2-ciLPs continued T cell lymphoid differentiation accompanied by c-KIT downregulation and interleukin 7 receptor expression. Switching off LMO2 expression was accompanied by increased Notch signaling and significant reduction of the CD44+CD25- myeloid cell population under T cell differentiation lymphoid conditions. Although vector insertions in cooperation with LMO2 expression could influence the fate of LMO2-ciLPs and additional experiments are required to evaluate it, our approach provides a promising tool to investigate mechanisms underlying stem cell, leukemia, and lymphocyte biology, leading to novel approaches for disease modeling and therapy evaluation.

Conditionally immortalized brown preadipocytes can switch between proliferative and differentiated states.

Brown adipose tissue (BAT) is a potential target to treat cardiometabolic disorders because of its capacity to combust glucose and fatty acids for thermoregulation. Its cellular and molecular investigation in humans is hampered by the limited availability of cell material and the heterogeneity of BAT between and within individuals. In this study, monoclonal lines of conditionally immortalized brown preadipocytes (iBPAs) of mouse and human origin were generated. Conditional immortalization was achieved by doxycycline-controlled expression of simian virus 40 large tumor antigen (LT) with a repressor-based Tet-On system. In the presence of doxycycline, both the murine and human cell lines showed long-term proliferation capacity with a population doubling time of ~28 h. After switching off LT expression by doxycycline removal and exposure to adipogenic differentiation medium, cells from both species acquired brown adipocyte properties. This was evidenced by the accumulation of multilocular lipid droplets, the upregulation of brown adipocyte markers including uncoupling protein 1 and an increase in lipolysis and oxygen consumption following adrenergic stimulation. Switching off LT expression before the onset of adipogenic differentiation was only critical for inducing adipogenesis in the human iBPAs, while their murine counterparts showed adipogenesis upon exposure to the adipogenic differentiation cocktail regardless of LT expression. When switched to proliferation medium, cultures of adipogenically differentiated human iBPAs de-differentiated and resumed cell division without losing their adipogenic capacity. We suggest that iBPAs represent an easy-to-use model for fundamental and applied research into BAT offering unique experimental opportunities due to their capacity to switch between proliferative and differentiated states.

A human beta cell line with drug inducible excision of immortalizing transgenes.

OBJECTIVES: Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-ßH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. METHODS: We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC ßH2 line. The resulting EndoC-ßH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. RESULTS: We showed that EndoC-ßH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 µg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. CONCLUSIONS: EndoC ßH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture.