Illuminating the Regenerative Properties of Stem Cells In Vivo with Bioluminescence Imaging

Preclinical animal studies are essential to the development of safe and effective stem cell therapies. Bioluminescence imaging (BLI) is a powerful tool in animal studies that enables the real-time longitudinal monitoring of stem cells in vivo to elucidate their regenerative properties. This review describes the application of BLI in preclinical stem cell research to address critical challenges in producing successful stem cell therapeutics. These challenges include stem cell survival, proliferation, homing, stress response, and differentiation. The applications presented here utilize bioluminescence to investigate a variety of stem and progenitor cells in several different in vivo models of disease and implantation. An overview of luciferase reporters is provided, along with the advantages and disadvantages of BLI. Additionally, BLI is compared to other preclinical imaging modalities and potential future applications of this technology are discussed in emerging areas of stem cell research.     

In Vivo Detection of Extrapancreatic Insulin Gene Expression in Diabetic Mice by Bioluminescence Imaging

Background

Extrapancreatic tissues such as liver may serve as potential sources of tissue for generating insulin-producing cells. The dynamics of insulin gene promoter activity in extrapancreatic tissues may be monitored in vivo by bioluminescence-imaging (BLI) of transgenic mice Tg(RIP-luc) expressing the firefly luciferase (luc) under a rat-insulin gene promoter (RIP).

Methods

The Tg(RIP-luc) mice were made diabetic by a single injection of the pancreatic β-cell toxin streptozotocin. Control mice were treated with saline. Mice were subject to serum glucose measurement and bioluminescence imaging daily. On day eight of the treatment, mice were sacrificed and tissues harvested for quantitative luciferase activity measurement, luciferase protein cellular localization, and insulin gene expression analysis.

Results

Streptozotocin-induced diabetic Tg(RIP-luc) mice demonstrated a dramatic decline in the BLI signal intensity in the pancreas and a concomitant progressive increase in the signal intensity in the liver. An average of 5.7 fold increase in the liver signal intensity was detected in the mice that were exposed to hyperglycemia for 8 days. Ex vivo quantitative assays demonstrated a 34-fold induction of the enzyme activity in the liver of streptozotocin-treated mice compared to that of the buffer-treated controls. Luciferase-positive cells with oval-cell-like morphology were detected by immunohistochemistry in the liver samples of diabetic mice, but not in that of non-treated control transgenic mice. Gene expression analyses of liver RNA confirmed an elevated expression of insulin genes in the liver tissue exposed to hyperglycemia.

Conclusions

BLI is a sensitive method for monitoring insulin gene expression in extrapancreatic tissues in vivo. The BLI system may be used for in vivo screening of biological events or pharmacologic activators that have the potential of stimulating the generation of extrapancreatic insulin-producing cells.     

In Vivo Fate Imaging of Intracerebral Stem Cell Grafts in Mouse Brain

We generated transgenic human neural stem cells (hNSCs) stably expressing the reporter genes Luciferase for bioluminescence imaging (BLI) and GFP for fluorescence imaging, for multimodal imaging investigations. These transgenic hNSCs were further labeled with a clinically approved perfluoropolyether to perform parallel ¹⁹F MRI studies. In vitro validation demonstrated normal cell proliferation and differentiation of the transgenic and additionally labeled hNSCs, closely the same as the wild type cell line, making them suitable for in vivo application. Labeled and unlabeled transgenic hNSCs were implanted into the striatum of mouse brain. The time profile of their cell fate after intracerebral grafting was monitored during nine days following implantation with our multimodal imaging approach, assessing both functional and anatomical condition. The ¹⁹F MRI demarcated the graft location and permitted to estimate the cell number in the graft. BLI showed a pronounce cell loss during this monitoring period, indicated by the decrease of the viability signal. The in vivo obtained cell fate results were further validated and confirmed by immunohistochemistry. We could show that the surviving cells of the graft continued to differentiate into early neurons, while the severe cell loss could be explained by an inflammatory reaction to the graft, showing the graft being surrounded by activated microglia and macrophages. These results are different from earlier cell survival studies of our group where we had implanted the identical cells into the same mouse strain but in the cortex and not in the striatum. The cortical transplanted cells did not show any loss in viability but only pronounced and continuous neuronal differentiation.     

V-Myc Immortalizes Human Neural Stem Cells in the Absence of Pluripotency-Associated Traits

A better understanding of the molecular mechanisms governing stem cell self-renewal will foster the use of different types of stem cells in disease modeling and cell therapy strategies. Immortalization, understood as the capacity for indefinite expansion, is needed for the generation of any cell line. In the case of v-myc immortalized multipotent human Neural Stem Cells (hNSCs), we hypothesized that v-myc immortalization could induce a more de-differentiated state in v-myc hNSC lines. To test this, we investigated the expression of surface, biochemical and genetic markers of stemness and pluripotency in v-myc immortalized and control hNSCs (primary precursors, that is, neurospheres) and compared these two cell types to human Embryonic Stem Cells (hESCs) and fibroblasts. Using a Hierarchical Clustering method and a Principal Component Analysis (PCA), the v-myc hNSCs associated with their counterparts hNSCs (in the absence of v-myc) and displayed a differential expression pattern when compared to hESCs. Moreover, the expression analysis of pluripotency markers suggested no evidence supporting a reprogramming-like process despite the increment in telomerase expression. In conclusion, v-myc expression in hNSC lines ensures self-renewal through the activation of some genes involved in the maintenance of stem cell properties in multipotent cells but does not alter the expression of key pluripotency-associated genes.     

In Vivo Volume and Hemoglobin Dynamics of Human Red Blood Cells

Human red blood cells (RBCs) lose ∼30% of their volume and ∼20% of their hemoglobin (Hb) content during their ∼100-day lifespan in the bloodstream. These observations are well-documented, but the mechanisms for these volume and hemoglobin loss events are not clear. RBCs shed hemoglobin-containing vesicles during their life in the circulation, and this process is thought to dominate the changes in the RBC physical characteristics occurring during maturation. We combine theory with single-cell measurements to investigate the impact of vesiculation on the reduction in volume, Hb mass, and membrane. We show that vesicle shedding alone is sufficient to explain membrane losses but not volume or Hb losses. We use dry mass measurements of human RBCs to validate the models and to propose that additional unknown mechanisms control volume and Hb reduction and are responsible for ∼90% of the observed reduction. RBC population characteristics are used in the clinic to monitor and diagnose a wide range of conditions including malnutrition, inflammation, and cancer. Quantitative characterization of cellular maturation processes may help in the early detection of clinical conditions where maturation patterns are altered.     

Graptopetalum Paraguayense Ameliorates Chemical-Induced Rat Hepatic Fibrosis In Vivo and Inactivates Stellate Cells and Kupffer Cells In Vitro

Background

Graptopetalum paraguayense (GP) is a folk herbal medicine with hepatoprotective effects that is used in Taiwan. The aim of this study was to evaluate the hepatoprotective and antifibrotic effects of GP on experimental hepatic fibrosis in both dimethylnitrosamine (DMN)- and carbon tetrachloride (CCl₄)-induced liver injury rats.

Methods

Hepatic fibrosis-induced rats were fed with the methanolic extract of GP (MGP) by oral administration every day. Immunohistochemistry, biochemical assays, and Western blot analysis were performed. The effects of MGP on the expression of fibrotic markers and cytokines in the primary cultured hepatic stellate cells (HSCs) and Kupffer cells, respectively, were evaluated.

Results

Oral administration of MGP significantly alleviated DMN- or CCl₄-induced liver inflammation and fibrosis. High levels of alanine transaminase, aspartate transaminase, bilirubin, prothrombin activity and mortality rates also decreased in rats treated with MGP. There were significantly decreased hydroxyproline levels in therapeutic rats compared with those of the liver-damaged rats. Collagen I and alpha smooth muscle actin (α-SMA) expression were all reduced by incubation with MGP in primary cultured rat HSCs. Furthermore, MGP induced apoptotic cell death in activated HSCs. MGP also suppressed lipopolysaccharide-stimulated rat Kupffer cell activation by decreasing nitric oxide, tumor necrosis factor-α and interleukin-6 production, and increasing interleukin-10 expression.

Conclusions

The results show that the administration of MGP attenuated toxin-induced hepatic damage and fibrosis in vivo and inhibited HSC and Kupffer cell activation in vitro, suggesting that MGP might be a promising complementary or alternative therapeutic agent for liver inflammation and fibrosis.     

In Vivo Imaging and Characterization of Actin Microridges

Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo.     

An Advanced Preclinical Mouse Model for Acute Myeloid Leukemia Using Patients' Cells of Various Genetic Subgroups and In Vivo Bioluminescence Imaging

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups.     

Survival of Neural Stem Cells Undergoing DNA Damage-Induced Astrocytic Differentiation in Self-Renewal-Promoting Conditions In Vitro

We recently reported that neural stem cells (NSCs) become senescent and commit to astrocytic differentiation upon X-ray irradiation. Surprisingly, under self-renewing culture conditions, some of these senescent cells undergo p53-independent apoptosis, which can be suppressed by caspase inhibition and BCL2 overexpression. Inhibition of apoptosis proved beneficial for astroglial differentiation efficiency; hence the toxicity of DNA damage on NSCs was specifically tested in context of the culture conditions. In this regard, self-renewal-promoting culture conditions proved incompatible with terminal astrocyte differentiation and impacted negatively on the viability of NSCs following DNA damage-induced cell cycle exit. On the contrary, a switch to differentiation-supporting conditions ablated apoptosis and conveyed tolerance to DNA damage. Thus, stem cell death has likely not originated from DNA break toxicity, while the potentially confounding effect of stem cell niche should always be taken in consideration in stem cell irradiation experiments.     

In Vivo 3D Meibography of the Human Eyelid Using Real Time Imaging Fourier-Domain OCT

Recently, we reported obtaining tomograms of meibomian glands from healthy volunteers using commercial anterior segment optical coherence tomography (AS-OCT), which is widely employed in clinics for examination of the anterior segment. However, we could not create 3D images of the meibomian glands, because the commercial OCT does not have a 3D reconstruction function. In this study we report the creation of 3D images of the meibomian glands by reconstructing the tomograms of these glands using high speed Fourier-Domain OCT (FD-OCT) developed in our laboratory. This research was jointly undertaken at the Department of Ophthalmology, Seoul St. Mary''s Hospital (Seoul, Korea) and the Advanced Photonics Research Institute of Gwangju Institute of Science and Technology (Gwangju, Korea) with two healthy volunteers and seven patients with meibomian gland dysfunction. A real time imaging FD-OCT system based on a high-speed wavelength swept laser was developed that had a spectral bandwidth of 100 nm at the 1310 nm center wavelength. The axial resolution was 5 µm and the lateral resolution was 13 µm in air. Using this device, the meibomian glands of nine subjects were examined. A series of tomograms from the upper eyelid measuring 5 mm (from left to right, B-scan) × 2 mm (from upper part to lower part, C-scan) were collected. Three-D images of the meibomian glands were then reconstructed using 3D “data visualization, analysis, and modeling software”. Established infrared meibography was also performed for comparison. The 3D images of healthy subjects clearly showed the meibomian glands, which looked similar to bunches of grapes. These results were consistent with previous infrared meibography results. The meibomian glands were parallel to each other, and the saccular acini were clearly visible. Here we report the successful production of 3D images of human meibomian glands by reconstructing tomograms of these glands with high speed FD-OCT.     

In Vitro and In Vivo Human Herpesvirus 8 Infection of Placenta

Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8) infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women.     

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades ¹. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective ^{2, 3}. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes ^{4, 5}. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease ^{6, 7}. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin ^{8, 9}. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.     

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells

In this study we used differentiated adult human upcyte^® cells for the in vitro generation of liver organoids. Upcyte^® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte^® process). Proliferating upcyte^® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte^® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.     

glpx Gene in Mycobacterium tuberculosis Is Required for In Vitro Gluconeogenic Growth and In Vivo Survival

Several enzymes involved in central carbon metabolism and gluconeogenesisplay a critical role in survival and pathogenesis of Mycobacterium tuberculosis (Mtb). The only known functional fructose 1,6-bisphosphatase (FBPase) in Mtb is encoded by the glpX gene and belongs to the Class II sub-family of FBPase. We describe herein the generation of a ΔglpX strain using homologous recombination. Although the growth profile of ΔglpX is comparable to that of wild type Mtb when grown on the standard enrichment media, its growth is dysgonic with individual gluconeogenic substrates such as oleic acid, glycerol and acetate. In mice lung CFU titers of ΔglpX were 2–3 log₁₀ lower than the wild-type Mtb strain. The results indicate that glpX gene encodes a functional FBPase and is essential for both in vitro and in vivo growth and survival of Mtb. Loss of glpX results in significant reduction of FBPase activity but not complete abolition. These findings verify that the glpX encoded FBPase II in Mtb can be a potential target for drug discovery.     

The Effects of Controlled Release of Neurotrophin-3 from PCLA Scaffolds on the Survival and Neuronal Differentiation of Transplanted Neural Stem Cells in a Rat Spinal Cord Injury Model

Neural stem cells (NSCs) have emerged as a potential source for cell replacement therapy following spinal cord injury (SCI). However, poor survival and low neuronal differentiation remain major obstacles to the use of NSCs. Biomaterials with neurotrophic factors are promising strategies for promoting the proliferation and differentiation of NSCs. Silk fibroin (SF) matrices were demonstrated to successfully deliver growth factors and preserve their potency. In this study, by incorporating NT-3 into a SF coating, we successfully developed NT-3-immobilized scaffolds (membranes and conduits). Sustained release of bioactive NT-3 from the conduits for up to 8 weeks was achieved. Cell viability was confirmed using live/dead staining after 14 days in culture. The efficacy of the immobilized NT-3 was confirmed by assessing NSC neuronal differentiation in vitro. NSC neuronal differentiation was 55.2±4.1% on the NT-3-immobilized membranes, which was significantly higher than that on the NT-3 free membrane. Furthermore, 8 weeks after the NSCs were seeded into conduits and implanted in rats with a transected SCI, the conduit+NT-3+NSCs group achieved higher NSC survival (75.8±15.1%) and neuronal differentiation (21.5±5.2%) compared with the conduit+NSCs group. The animals that received the conduit+NT-3+NSCs treatment also showed improved functional outcomes, as well as increased axonal regeneration. These results indicate the feasibility of fabricating NT-3-immobilized scaffolds using the adsorption of NT-3/SF coating method, as well as the potential of these scaffolds to induce SCI repair by promoting survival and neuronal differentiation of transplanted NSCs.