A Humanized Patient-Derived Xenograft Model for Pancreatic Cancer.

Pancreatic cancer is associated with a high mortality rate, and there are still very few effective treatment options. Patient-derived xenografts have proven to be invaluable preclinical disease models to study cancer biology and facilitate testing of novel therapeutics. However, the severely immune-deficient mice used to generate standard models lack any functional immune system, thereby limiting their utility as a tool to investigate the tumor-immune cell interface. This chapter will outline a method for establishment of "humanized" patient-derived xenografts, which are reconstituted with human immune cells to imitate the immune-rich microenvironment of pancreatic cancer.

Endoscopic Ultrasound-Guided Fine-Needle Biopsies to Generate Preclinical Disease Models to Study Inflammation in Pancreatic Ductal Adenocarcinoma.

Patient-derived xenografts (PDXs) are valuable models to study cancer biology, behavior, and response to therapies in vivo. Pancreatic cancer is an aggressive and treatment-resistant disease, and typical biopsies are often of low cellular yield and therefore present challenges for the creation of PDXs. This chapter will describe a method to establish PDX models from tissue biopsies obtained via endoscopic ultrasound-guided fine-needle aspiration, a relatively noninvasive technique which compared to surgery is available to pancreatic cancer patients at all stages of disease. Furthermore, we also describe methods to incorporate "humanization" of PDXs via reconstitution with human immune cells, thus mimicking the immune cell-rich microenvironment of pancreatic tumors.

A Mitochondrion-Targeting Protein (B2) Primes ROS/Nrf2-Mediated Stress Signals, Triggering Apoptosis and Necroptosis in Lung Cancer.

The betanodavirus B2 protein targets mitochondria and triggers mitochondrion-mediated cell death signaling in lung cancer cells; however, its molecular mechanism remains unknown. In this study, we observed that B2 triggers hydrogen peroxide/Nrf2-involved stress signals in the dynamic regulation of non-small lung cancer cell (NSCLC)-programmed cell death. Here, the B2 protein works as a necrotic inducer that triggers lung cancer death via p53 upregulation and RIP3 expression, suggesting a new perspective on lung cancer therapy. We employed the B2 protein to target A549 lung cancer cells and solid tumors in NOD/SCID mice. Tumors were collected and processed for the hematoxylin and eosin staining of tissue and cell sections, and their sera were used for blood biochemistry analysis. We observed that B2 killed an A549 cell-induced solid tumor in NOD/SCID mice; however, the mutant ΔB2 did not. In NOD/SCID mice, B2 (but not ΔB2) induced both p53/Bax-mediated apoptosis and RIPK3-mediated necroptosis. Finally, immunochemistry analysis showed hydrogen peroxide /p38/Nrf2 stress strongly inhibited the production of tumor markers CD133, Thy1, and napsin, which correlate with migration and invasion in cancer cells. This B2-triggered, ROS/Nrf2-mediated stress signal triggered multiple signals via pathways that killed A549 lung cancer tumor cells in vivo. Our results provide novel insight into lung cancer management and drug therapy.

Conjugation of Diclofenac with Novel Oleanolic Acid Derivatives Modulate Nrf2 and NF-κB Activity in Hepatic Cancer Cells and Normal Hepatocytes Leading to Enhancement of Its Therapeutic and Chemopreventive Potential.

Combining NSAIDs with conventional therapeutics was recently explored as a new strategy in cancer therapy. Our earlier studies showed that novel oleanolic acid oximes (OAO) conjugated with aspirin or indomethacin may enhance their anti-cancer potential through modulation of the Nrf2 and NF-κB signaling pathways. This study focused on the synthesis and biological evaluation of four diclofenac (DCL)-OAO derivative conjugates in the context of these pathways' modification and hepatic cells survival. Treatment with the conjugates 4d, 3-diclofenacoxyiminoolean-12-en-28-oic acid morpholide, and 4c, 3-diclofenacoxyiminoolean-12-en-28-oic acid benzyl ester significantly reduced cell viability in comparison to the DCL alone. In THLE-2, immortalized normal hepatocytes treated with these conjugates resulted in the activation of Nrf2 and increased expression in SOD-1 and NQO1, while the opposite effect was observed in the HepG2 hepatoma cells. In both cell lines, reduced activation of the NF-κB and COX-2 expression was observed. In HepG2 cells, conjugates increased ROS production resulting from a reduced antioxidant defense, induced apoptosis, and inhibited cell proliferation. In addition, the OAO morpholide derivative and its DCL hybrid reduced the tumor volume in mice bearing xenografts. In conclusion, our study demonstrated that conjugating diclofenac with the OAO morpholide and a benzyl ester might enhance its anti-cancer activity in HCC.

Lentinan inhibited colon cancer growth by inducing endoplasmic reticulum stress-mediated autophagic cell death and apoptosis.

Lentinan (SLNT) has been shown to be directly cytotoxic to cancer cells. However, this direct antitumour effect has not been thoroughly investigated in vivo, and the mechanism remains unclear. We aimed to examine the direct antitumour effect of SLNT on human colon cancer and the mechanism in vivo and in vitro. SLNT significantly inhibited tumour growth and induced autophagy and endoplasmic reticulum stress (ERS) in HT-29 cells and tumour-bearing nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. Experiments with the autophagy inhibitors chloroquine (CQ) and 3-methyladenine (3-MA) showed that autophagy facilitated the antitumour effect of SLNT. Moreover, ERS was identified as the common upstream regulator of SLNT-induced increases in Ca2+concentrations, autophagy and apoptosis by using ERS inhibitors. In summary, our study demonstrated that SLNT exerted direct antitumour effects on human colon cancer via ERS-mediated autophagy and apoptosis, providing a novel understanding of SLNT as an anti-colon cancer therapy.

Anticancer activities of parthenolide in primary effusion lymphoma preclinical models.

The sesquiterpene lactone parthenolide is a major component of the feverfew medicinal plant, Tanacetum parthenium. Parthenolide has been extensively studied for its anti-inflammatory and anticancer properties in several tumor models. Parthenolide's antitumor activities depend on several mechanisms but it is mainly known as an inhibitor of the nuclear factor-κB (NF-κB) pathway. This pathway is constitutively activated and induces cell survival in primary effusion lymphoma (PEL), a rare aggressive AIDS-related lymphoproliferative disorder that is commonly caused by the human herpesvirus 8 (HHV-8) infection. The aim of this study is to evaluate the targeted effect of Parthenolide both in vitro and in vivo. Herein, parthenolide significantly inhibited cell growth, induced G0 /G1 cell cycle arrest, and induced massive apoptosis in PEL cells and ascites. In addition, parthenolide inhibited the NF-ĸB pathway suppressing IĸB phosphorylation and p65 nuclear translocation. It also reduced the expression of the DNA methylase inhibitor (DNMT1). Parthenolide induced HHV-8 lytic gene expression without inhibiting latent viral gene expression. Importantly, DMAPT, the more soluble parthenolide prodrug, promoted delay in ascites development and prolonged the survival of PEL xenograft mice. This study supports the therapeutic use of parthenolide in PEL and encourages its further clinical development.

Development of Cutaneous Wound in Diabetic Immunocompromised Mice and Use of Dental Pulp-Derived Stem Cell Product for Healing.

Chronic nonhealing wounds impact nearly 15% of Medicare beneficiaries (8.2 million) in the United States costing $28-$32 billion annually. Despite advancement in wound management, approximately 8% of diabetic Medicare beneficiaries have a foot ulcer and 1.8% will have an amputation. The development of a regenerative approach is warranted to save these before-mentioned amputations. To this extent, herein, we describe the detailed methods in generating a type 1 diabetes mellitus (T1DM) condition in immunocompromised mice, inducing cutaneous wound, and application of dental pulp stem cell-derived secretory products for therapeutic assessment. This model helps in evaluating the efficacy of stem cell-based therapy and helps with the investigation of involved mechanisms in impaired cutaneous wound healing caused by hyperglycemic stress due to type 1 diabetes.

Cutaneous Wound Generation in Diabetic NOD/SCID Mice and the Use of Nanofiber-Expanded Hematopoietic Stem Cell Therapy.

Despite significant advances in diabetic wound management, diabetic wounds remain a significant global problem that decreases patient's quality of life, and chronic wounds may lead to amputation and death to the patients. To develop a potential regenerative therapy, a xenogeneic transplantation compatible laboratory model needs to be developed. This procedure demonstrates how to isolate hematopoietic stem cells (CD133+) from human umbilical cord blood, expand CD34+ stem cells using a nanofiber scaffold (polyether sulfone-coated and amino group-treated), induce diabetes in immunocompromised (NOD/SCID) mice, induce a cutaneous wound in mice, and how to treat the wound with the nanofiber-expanded CD34+ stem cells. This protocol also shows how to measure wound healing.

BRAF V600E mutation and MET amplification as resistance pathways of the second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib in lung cancer.

BACKGROUND: Anaplastic lymphoma kinase (ALK) targeted therapies have demonstrated remarkable efficacy in ALK-positive lung adenocarcinomas. However, patients inevitably develop resistance to such therapies. To investigate novel mechanisms of resistance to second generation ALK inhibitors, we characterized and modeled ALK inhibitor resistance of ALK-positive patient-derived xenograft (PDX) models established from advanced-stage lung adenocarcinoma patients who have progressed on one or more ALK inhibitors. METHODS: Whole exome sequencing was performed to identify resistance mechanisms to ALK inhibitors in PDXs generated from biopsies at the time of relapse. ALK fusion status was confirmed using fluorescent in situ hybridization, immunohistochemistry, RNA-sequencing, RT-qPCR and western blot. Targeted therapies to overcome acquired resistance were then tested on the PDX models. RESULTS: Three PDX models were successfully established from biopsies of two patients who had progressed on crizotinib and/or alectinib. The PDX models recapitulated the histology and ALK status of their patient tumors, as well as their matched patients' clinical treatment outcome to ALK inhibitors. Whole exome sequencing identified MET amplification and previously unreported BRAF V600E mutation as independent mechanisms of resistance to alectinib. Importantly, PDX treatment of inhibitors specific for these targets combined with ALK inhibitor overcame resistance. CONCLUSIONS: Bypass signaling pathway through c-MET and BRAF are independent mechanisms of resistance to alectinib. Individualized intervention against these resistance pathways could be viable therapeutic options in alectinib-refractory lung adenocarcinoma.

Spontaneous murine tumors in the development of patient-derived xenografts: a potential pitfall.

Patient-derived xenografts (PDX) are generated in immune deficient mice and demonstrate histologic and molecular features similar to their corresponding human tumors. However, murine tumors (non-human) spontaneously occur in these models. 120 consecutive patients with high-risk primary breast cancer enrolled in the prospective neoadjuvant BEAUTY study had tumor tissue obtained at the time of diagnosis. These tumor cells, including initial tissue and subsequent generations, were injected into either NSG (n = 365) or NOD-SCID (n = 396) female mice. Mice with initial tumor growth sufficient for transfer to the 2nd generation underwent histologic review by pathologists, including Ki67 staining. After passaging the tumors for up to 4 generations, at least one primary mouse tumor was detected from 24 of the 54 PDX-lines, for a frequency of 3.2% (24 mice out of 761 mice), including murine lymphomas (n = 13), mammary tumors (n = 7), osteosarcomas (n = 2), and hemangiosarcomas (n = 2). While true PDX showed scattered strong staining with Ki67, murine tumors were Ki67 negative. No significant differences (p = 0.062) were observed comparing development of murine tumors in NOD-SCID (n = 8) vs NSG mice (n = 16). While PDX are a useful tool in cancer research, there is a potential for spontaneous murine tumors to arise, which could alter results of studies utilizing PDX. Morphologic review by a pathologist, potentially along with Ki67 staining, is necessary to ensure that tumor growth represents the desired PDX prior to use in downstream studies. This study is the first prospective study evaluating the frequency, type, and time frame for development of non-human tumors.

Safety and efficacy of ex vivo expanded CD34+ stem cells in murine and primate models.

BACKGROUND: Hematopoietic stem cell (HSC) transplantation has been widely applied to the treatment of malignant blood diseases. However, limited number of functional HSCs hinders successful transplantation. The purpose of our current study is to develop a new and cost-efficient medium formulation that could greatly enhance the expansion of HSCs while retaining their long-term repopulation and hematopoietic properties for effective clinical transplantation. METHODS: Enriched human CD34+ cells and mobilized nonhuman primate peripheral blood CD34+ cells were expanded with a new, cost-efficient expansion medium formulation, named hematopoietic expansion medium (HEM), consisting of various cytokines and nutritional supplements. The long-term repopulation potential and hematologic-lineage differentiation ability of expanded human cells were studied in the non-obese diabetic/severe combined immunodeficiency mouse model. Furthermore, the efficacy and safety studies were performed by autologous transplantation of expanded primate cells in the nonhuman primate model. RESULTS: HEM could effectively expand human CD34+ cells by up to 129 fold within 9 days. Expanded HSCs retained long-term repopulation potential and hematologic-lineage differentiation ability, as indicated by (1) maintenance (over unexpanded HSCs) of immunophenotypes of CD38-CD90+CD45RA-CD49f+ in CD34+ cells after expansion; (2) significant presence of multiple human hematopoietic lineages in mouse peripheral blood and bone marrow following primary transplantation; (3) enrichment (over unexpanded HSCs) in SCID-repopulating cell frequency measured by limiting dilution analysis; and (4) preservation of both myeloid and lymphoid potential among human leukocytes from mouse bone marrow in week 24 after primary transplantation or secondary transplantation. Moreover, the results of autologous transplantation in nonhuman primates demonstrated that HEM-expanded CD34+ cells could enhance hematological recovery after myelo-suppression. All primates transplanted with the expanded autologous CD34+ cells survived for over 18 months without any noticeable abnormalities. CONCLUSIONS: Together, these findings demonstrate promising potential for the utility of HEM to improve expansion of HSCs for clinical application.

Comparison between NOD/SCID mice and BALB/c mice for patient-derived tumor xenografts model of non-small-cell lung cancer.

BACKGROUND: Patient-derived tumor xenografts (PDX) are considered as a more reliable experiment model for screening chemotherapeutic drugs. However, the tumorigenic rate differs depending on mouse strains, which generates the experimental variability. MATERIALS AND METHODS: In this study, we built PDX models of human non-small-cell lung cancer (NSCLC) in NOD/SCID mice in comparison with BALB/c mice. RESULTS: The result showed that the tumorigenesis rate of NOD/SCID mice (46.2%, 18/39) was higher than that of BALB/c mice (17.39%, 4/23). Latent times of tumorigenesis of NOD/SCID mice (41±18 days) were shorter than these of BALB/c mice (53±17 days). Times of tumorigenesis of NOD/SCID mice (85±25 days) were shorter than that of BALB/c mice (104±14 days). In addition, squamous carcinoma tissues were more likely to form tumors than adenocarcinoma tissues in NOD/SCID mice (P=0.008) and BALB/c mice (P=0.09). Also tumors could retain patients' tumor characteristics in NOD/SCID mice and BALB/c mice xenograft models. CONCLUSION: It is worth mentioning that the result of the drug experiment in the PDX models was consistent with the effect of clinical chemotherapy. As a result, NOD/SCID mice have advantages in a higher rate of tumorigenesis, shorter latent times of tumorigenesis and times of tumorigenesis over BALB/c mice in PDX models. It can provide a more reliable model of drug screening.

STAT3 Knockdown Induces Tumor Formation by MDA-MB-231 Cells.

STAT3 plays a central role in oncogenesis by mediating cell survival, growth, and differentiation. It is constitutively activated in breast cancer. We investigated the role of STAT3 in tumor development by knocking down STAT3 levels in MDA-MB-231 triple negative breast cancer cells using short hairpin RNA. The tumor forming potential of these STAT3-depleted cells was assessed by xenografts in immunocompromised NOD SCID mice. Contrary to its accepted tumor promoting role, we found STAT3 to be a negative regulator of growth in MDA-MB-231- derived tumors. Although similar observations have been made in thyroid carcinoma and lung adenocarcinoma xenograft studies, our novel results showed for the first time that the role of STAT3 in promoting tumorigenesis may be context-specific, and that STAT3 may actually be a negative regulator of certain breast-cancer types. Studies to identify the mechanisms of STAT3's negative regulatory role may be useful in developing STAT3-based therapeutics.

Utility of Endoscopic Ultrasound-Guided Fine-Needle Aspiration for Preclinical Evaluation of Therapies in Cancer.

Personalising cancer therapy is a way of improving treatment efficacy, by selecting specific treatments for patients with certain molecular changes to their tumour. This requires both molecular material to detect these targets and a preclinical disease model to demonstrate treatment efficacy. In pancreatic cancer this is problematic, as most patients present with advanced disease and are therefore ineligible for surgery. As a result, biological material derived from such patients has been excluded from all preclinical studies in personalised medicine. This chapter presents methodology to achieve both of the above-mentioned requirements using endoscopic ultrasound-guided fine-needle aspiration, which can be offered to nearly all patients with early or advanced disease.

Tumour-associated glial host cells display a stem-like phenotype with a distinct gene expression profile and promote growth of GBM xenografts.

BACKGROUND: Little is known about the role of glial host cells in brain tumours. However, supporting stromal cells have been shown to foster tumour growth in other cancers. METHODS: We isolated stromal cells from patient-derived glioblastoma (GBM) xenografts established in GFP-NOD/scid mice. With simultaneous removal of CD11b+ immune and CD31+ endothelial cells by fluorescence activated cell sorting (FACS), we obtained a population of tumour-associated glial cells, TAGs, expressing markers of terminally differentiaed glial cell types or glial progenitors. This cell population was subsequently characterised using gene expression analyses and immunocytochemistry. Furthermore, sphere formation was assessed in vitro and their glioma growth-promoting ability was examined in vivo. Finally, the expression of TAG related markers was validated in human GBMs. RESULTS: TAGs were highly enriched for the expression of glial cell proteins including GFAP and myelin basic protein (MBP), and immature markers such as Nestin and O4. A fraction of TAGs displayed sphere formation in stem cell medium. Moreover, TAGs promoted brain tumour growth in vivo when co-implanted with glioma cells, compared to implanting only glioma cells, or glioma cells and unconditioned glial cells from mice without tumours. Genome-wide microarray analysis of TAGs showed an expression profile distinct from glial cells from healthy mice brains. Notably, TAGs upregulated genes associated with immature cell types and self-renewal, including Pou3f2 and Sox2. In addition, TAGs from highly angiogenic tumours showed upregulation of angiogenic factors, including Vegf and Angiopoietin 2. Immunohistochemistry of three GBMs, two patient biopsies and one GBM xenograft, confirmed that the expression of these genes was mainly confined to TAGs in the tumour bed. Furthermore, their expression profiles displayed a significant overlap with gene clusters defining prognostic subclasses of human GBMs. CONCLUSIONS: Our data demonstrate that glial host cells in brain tumours are functionally distinct from glial cells of healthy mice brains. Furthermore, TAGs display a gene expression profile with enrichment for genes related to stem cells, immature cell types and developmental processes. Future studies are needed to delineate the biological mechanisms regulating the brain tumour-host interplay.

An effective ex-vivo approach for inducing endothelial progenitor cells from umbilical cord blood CD34+ cells.

BACKGROUND: Transplantation of endothelial progenitor cells (EPCs)/endothelial cells (ECs) has been used for the treatment of ischemic diseases and hemophilia A, due to their great capacity for producing factor VIII and for repairing vascular damage. We established an effective approach to stimulate the expansion and differentiation of EPCs for potential therapeutic applications. METHODS: CD34+ cells isolated from human cord blood were cultured in a two-step system for 21 days. The generated adherent cells were characterized via flow cytometry and immunofluorescent staining. Moreover, single-cell clonogenic and tube-forming assays were carried out to evaluate their potential to proliferate and form vessel networks. Furthermore, these cells were transplanted into a mouse model of hepatic sinusoidal endothelium injury by hepatic portal vein injection to investigate their in-vivo behavior. RESULTS: The two-step culture protocol promoted the expansion and differentiation of human cord blood CD34+ cells efficiently, resulting in a large number of adherent cells within 3 weeks. The generated adherent cells were identified as EPCs/ECs based on the expression of CD31, CD144, vWF, and FVIII, and cell numbers showed a 1400-fold increase compared with the initial number. Moreover, these EPCs/ECs were capable of proliferating and establishing colonies as individual cells, and forming tube-like structures. More significantly, tissue examination of mice after transplantation revealed that the injected EPCs/ECs migrated and integrated into the liver, reconstituting the sinusoidal endothelial compartment. CONCLUSIONS: We developed an approach for the generation of cord blood-derived EPCs/ECs on a large scale, characterized them phenotypically, and demonstrated their in-vivo functional capacity. Our approach provides an excellent source of healthy EPCs/ECs for use in cell therapy in a clinical setting.

Teratoma Formation Assay for Assessing Pluripotency and Tumorigenicity of Pluripotent Stem Cells.

Pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) form teratomas when transplanted into immunodeficient mice. As teratomas contain all three germ layers (endoderm, mesoderm, ectoderm), teratoma formation assay is widely used as an index of pluripotency (Evans and Kaufman, 1981; Hentze et al., 2009 ; Gropp et al., 2012 ). On the other hand, teratoma-forming tumorigenicity also represents a major risk factor impeding potential clinical applications of pluripotent stem cells ( Miura et al., 2009 ; Okano et al., 2013 ). Recently, we reported that iPSCs derived from naked mole-rat lack teratoma-forming tumorigenicity when engrafted into the testes of non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice due to an ES cell-expressed Ras (ERAS) and Alternative reading frame (ARF)-dependent tumor-suppression mechanism specific to this species ( Miyawaki et al., 2016 ). Here, we describe a method for transplanting pluripotent stem cells into the testes of NOD/SCID mice to generate teratomas for assessing the pluripotency and tumorigenicity.

NOD-SCID mice irradiation with medical accelerators: Dosimetric and radiobiological results.

PURPOSE: Preclinical studies normally requires dedicated instruments due to the small anatomical scales involved, but the possibility of using clinical devices for this purpose may be of economical, scientific and translational interest. In the present work the accurate description of treatment planning, dosimetric results, radiotoxicity and tumor response of the irradiation of NOD-SCID mice were presented. Two medical linear accelerators, TrueBeam STx and Tomotherapy Hi-ART, were compared. NOD-SCID mice irradiation with Tomotherapy is a novelty, as well as the comparison of different irradiation techniques, devices and dose fractionations. METHODS: Human derived glioblastoma multiforme neurospheres were injected in immunocompromised NOD-SCID mice to establish xenograft models. Mice were anaesthetized and placed in a plexiglas cage pieboth to perform CT scan for treatment planning purposes and for the irradiation. Three fractionation schedules were evaluated: 4Gy/1 fraction, 4Gy/2 fractions and 6Gy/3 fractions. Tomotherapy planning parameters, the presence of a bolus layer and the irradiation time were reported. After irradiation, mice were examined daily and sacrificed when they showed signs of suffering or when tumor volume reached the established endpoint. Outcomes regarding both radiotoxicity and tumor response were evaluated comparing irradiated mice as respect to their controls. RESULTS: Survival analysis showed that Tomotherapy irradiation with 6Gy/3 fractions with a bolus layer prolong mice survival (log-rank test, p<0.02). Tumor volume and mice survival were significantly different in irradiated xenografts as compared to their controls (t-test, p<0.03; log-rank, p<0.05). CONCLUSION: The radiobiological potential of Tomotherapy in inducing tumor growth stabilization is demonstrated.

Ex-vivo expansion of nonhuman primate CD34+ cells by stem cell factor Sall4B.

BACKGROUND: Hematopoietic CD34+ stem cells are widely used in the clinical therapy of complicated blood diseases. Stem cell factor Sall4B is a zinc finger transcription factor that plays a vital role in hematopoietic stem cell expansion. The purpose of our current study is to further evaluate how Sall4B might affect the expansion of CD34+ cells derived from nonhuman primates. METHODS: Sall4B was overexpressed in nonhuman primate bone marrow-derived CD34+ cells via a lentiviral transduction system. The granulocyte-erythrocyte-macrophage-megakaryocyte colony-forming unit (CFU) assay evaluated the differentiation potential of primate CD34+ cells that were expanded with Sall4B. Furthermore, an in-vivo murine system was employed to evaluate the hematopoietic potential of primate Sall4B-expanded CD34+ cells. RESULTS: Overexpression of Sall4B promoted ex-vivo nonhuman primate CD34+ cell expansion by 9.21 ± 1.94-fold on day 9, whereas lentiviral transduction without Sall4B expanded cells by only 2.95 ± 0.77-fold. Sall4B maintained a significant percentage of CD34+ cells as well. The CFU assay showed that the Sall4B-expanded CD34+ cells still possessed multilineage differentiation potential. A study using nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice in vivo revealed that Sall4B led to an increase in the number of repopulating cells and the 9-day-old Sall4B-transduced CD34+ cells still possess self-renewal and multilineage differentiation capacity in vivo, which are similar stemness characteristics to those in freshly isolated primate bone marrow-derived CD34+ cells. CONCLUSIONS: We investigated the expansion of nonhuman primate bone marrow-derived CD34+ cells using the Sall4B lentiviral overexpression approach; our findings provide a new perspective on mechanisms of rapid stem cell proliferation. The utilization of Sall4B to expand CD34+ cells on a large scale through use of suitable model systems would prove helpful towards preclinical trials of autologous transplantation.

Relative distribution of Gb3 isoforms/analogs in NOD/SCID/Fabry mice tissues determined by tandem mass spectrometry.

AIM: Fabry disease is a lysosomal storage disorder leading to glycosphingolipid accumulation in different organs, tissues and biological fluids. The development of a Fabry disease gene therapy trial is underway in Canada. A tool to determine the distribution of Gb3 biomarkers in tissues of Fabry mice might be applicable to monitor the effect of gene therapy. Results & methodology: An ultra-performance LC-MS/MS (UPLC-MS/MS) method for the analysis of 22 Gb3 isoform/analogs in various Fabry mice tissues was developed and validated. Marked variation in biomarker organ distribution was found with higher levels in the spleen, followed by the small intestine, kidneys, lungs, heart, liver and brain. CONCLUSION: The devised method is sensitive and useful for the evaluation of biomarker profiles in Fabry mice.