Cell Lineage Identification and Stem Cell Culture in a Porcine Model for the Study of Intestinal Epithelial Regeneration

Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC) driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM) and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA), Minichromosome Maintenance Complex 2 (MCM2), Bromodeoxyuridine (BrdU) and phosphorylated Histone H3 (pH3) distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/‘reserve’ stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2); enteroendocrine cells by Chromogranin A (CGA), Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII) and sucrase isomaltase (SIM). Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease.     

A Novel Zebrafish Xenotransplantation Model for Study of Glioma Stem Cell Invasion

Invasion and metastasis of solid tumors are the major causes of death in cancer patients. Cancer stem cells (CSCs) constitute a small fraction of tumor cell population, but play a critical role in tumor invasion and metastasis. The xenograft of tumor cells in immunodeficient mice is one of commonly used in vivo models to study the invasion and metastasis of cancer cells. However, this model is time-consuming and labor intensive. Zebrafish (Danio rerio) and their transparent embryos are emerging as a promising xenograft tumor model system for studies of tumor invasion. In this study, we established a tumor invasion model by using zebrafish embryo xenografted with human glioblastoma cell line U87 and its derived cancer stem cells (CSCs). We found that CSCs-enriched from U87 cells spreaded via the vessels within zebrafish embryos and such cells displayed an extremely high level of invasiveness which was associated with the up-regulated MMP-9 by CSCs. The invasion of glioma CSCs (GSCs) in zebrafish embryos was markedly inhibited by an MMP-9 inhibitor. Thus, our zebrafish embryo model is considered a cost-effective approach tostudies of the mechanisms underlying the invasion of CSCs and suitable for high-throughput screening of novel anti-tumor invasion/metastasis agents.     

Successful Fertility Restoration after Allogenic Hematopoietic Stem Cell Transplantation

ObjectiveMyeloablative conditioning regimens given prior to hematopoietic stem cell transplantation (HSCT) frequently cause permanent sterility in men. In patients with sickle cell disease (SCD) we use a nonmyeloablative regimen with sirolimus, alemtuzumab, and low-dose total-body irradiation (300 centigrays) with gonadal shielding preceding allogeneic HSCT. We report here the restoration of azoospermia in a patient with SCD after allogeneic HSCT. We discuss the impact of our patient’s underlying chronic medical conditions and the therapies he had received (frequent blood transfusions, iron chelating drugs, ribavirin, hydroxyurea, opioids), as well as the impact of the nonmyeloablative conditioning regimen on male gonadal function, and we review the literature on this topic.MethodsWe determined the patient’s reproductive hormonal values and his semen parameters before, during, and after HSCT and infertility treatment. In addition, we routinely measured his serum laboratory parameters pertinent to SCD and infertility, such as iron and ferritin levels. A karyotype analysis was performed to assess the potential presence of Klinefelter syndrome. Finally, imaging studies of the patient’s brain and testes were done to rule out further underlying pathology.ResultsA 42-year-old man with SCD, transfusional iron overload, and hepatitis C underwent a nonmyeloablative allogeneic HSCT. One year later he desired to father a child but was found to be azoospermic in the context of hypogonadotropic hypogonadism. Restoration of fertility was attempted with human chorionic gonadotropin (2,000 IU) plus human menopausal gonadotropin (75 IU follicle-stimulating hormone) injected subcutaneously 3 times weekly. Within 6 months of treatment, the patient’s serum calculated free testosterone value normalized, and his sperm count and sperm motility improved. After 10 months, he successfully initiated a pregnancy through intercourse. The pregnancy was uncomplicated, and a healthy daughter was delivered naturally at term.ConclusionDespite exposure to several gonadotoxins, transfusional iron overload and nonmyeloablative conditioning with radiation causing severe testicular atrophy suggesting extensive damage to seminiferous tubules and possibly Leydig cells, gonadotropins were efficacious in restoring our patient’s reproductive capability. (Endocr Pract. 2014;20:e157-e161)     

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors ^1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., {"type":"clinical-trial","attrs":{"text":"NCT01210911","term_id":"NCT01210911"}}NCT01210911, {"type":"clinical-trial","attrs":{"text":"NCT01167738","term_id":"NCT01167738"}}NCT01167738, and {"type":"clinical-trial","attrs":{"text":"NCT01488552","term_id":"NCT01488552"}}NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.     

Enteric Neurospheres Are Not Specific to Neural Crest Cultures: Implications for Neural Stem Cell Therapies

Objectives

Enteric neural stem cells provide hope of curative treatment for enteric neuropathies. Current protocols for their harvesting from humans focus on the generation of ‘neurospheres’ from cultures of dissociated gut tissue. The study aims to better understand the derivation, generation and composition of enteric neurospheres.

Design

Gut tissue was obtained from Wnt1-Cre;Rosa26^Yfp/Yfp transgenic mice (constitutively labeled neural crest cells) and paediatric patients. Gut cells were cultured either unsorted (mixed neural crest/non-neural crest), or following FACS selection into neural crest (murine-YFP+ve/human-p75+ve) or non-neural crest (YFP-ve/p75-ve) populations. Cultures and resultant neurospheres were characterized using immunolabelling in vitro and following transplantation in vivo.

Results

Cultures of (i) unsorted, (ii) neural crest, and (iii) non-neural crest cell populations generated neurospheres similar in numbers, size and morphology. Unsorted neurospheres were highly heterogeneous for neural crest content. Neural crest-derived (YFP+ve/p75+ve) neurospheres contained only neural derivatives (neurons and glia) and were devoid of non-neural cells (i.e. negative for SMA, c-Kit), with the converse true for non-neural crest-derived (YFP-ve/p75-ve) ‘neurospheres’. Under differentiation conditions only YFP+ve cells gave rise to neural derivatives. Both YFP+ve and YFP-ve cells displayed proliferation and spread upon transplantation in vivo, but YFP-ve cells did not locate or integrate within the host ENS.

Conclusions

Spherical accumulations of cells, so-called ‘neurospheres’ forming in cultures of dissociated gut contain variable proportions of neural crest-derived cells. If they are to be used for ENS cell replacement therapy then improved protocols for their generation, including cell selection, should be sought in order to avoid inadvertent transplantation of non-therapeutic, non-ENS cells.     

利用胚胎干细胞分化的拟胚体研究小鼠早期胚胎发育过程

小鼠胚胎干细胞(ES细胞)具有分化的全能性已经得到广泛共识。ES细胞在体外分化所形成的拟胚体在结构上能够模仿早期胚胎发育过程,包括在内细胞团表面形成内胚层、柱状上皮细胞的分化,以及中央空腔的形成。本文介绍利用拟胚体研究小鼠早期胚胎发育过程中各个胚胎阶段的发育、细胞程序性死亡的发生及TGF-β信号在胚胎发育过程中的作用。     

Establishment of a Murine Graft-versus-Myeloma Model Using Allogeneic Stem Cell Transplantation

BackgroundMultiple myeloma (MM) is a malignant plasma cell disorder with poor long-term survival and high recurrence rates. Despite evidence of graft-versus-myeloma (GvM) effects, the use of allogeneic hematopoietic stem cell transplantation (allo-SCT) remains controversial in MM. In the current study, we investigated the anti-myeloma effects of allo-SCT from B10.D2 mice into MHC-matched myeloma-bearing Balb/cJ mice, with concomitant development of chronic graft-versus-host disease (GvHD).ConclusionsWe successfully established an immunocompetent murine model of graft-versus-myeloma. This is the first murine GvM model using immunocompetent mice that develop MM which closely resembles human MM disease and that are treated after disease establishment with an allo-SCT. Importantly, using TCR Vβ spectratyping, we also demonstrated the presence of GvM unique responses potentially associated with the curative capacity of this immunotherapeutic approach.     

重组人干细胞因子的研究

利用基因工程的方法制备了高纯度的人干细胞因子,并对制备的重组人干细胞因子的结构和生物活性进行了研究。结果表明rhSCF在磷酸盐缓冲液中以非共价的二聚体形式存在,其精确分子量、质量肽谱、氨基酸组成及N端、C段序列等均与理论值一致,二硫键位置正确。rhSCF单用或与rhG-CSF合用,对猴外周血造血祖细胞有明显的动员作用。     

Non-Invasive Stem Cell Therapy in a Rat Model for Retinal Degeneration and Vascular Pathology

Background

Retinitis pigmentosa (RP) is characterized by progressive night blindness, visual field loss, altered vascular permeability and loss of central vision. Currently there is no effective treatment available except gene replacement therapy has shown promise in a few patients with specific gene defects. There is an urgent need to develop therapies that offer generic neuro-and vascular-protective effects with non-invasive intervention. Here we explored the potential of systemic administration of pluripotent bone marrow-derived mesenchymal stem cells (MSCs) to rescue vision and associated vascular pathology in the Royal College Surgeons (RCS) rat, a well-established animal model for RP.

Methodology/Principal Findings

Animals received syngeneic MSCs (1×10⁶ cells) by tail vein at an age before major photoreceptor loss. Principal results: both rod and cone photoreceptors were preserved (5–6 cells thick) at the time when control animal has a single layer of photoreceptors remained; Visual function was significantly preserved compared with controls as determined by visual acuity and luminance threshold recording from the superior colliculus; The number of pathological vascular complexes (abnormal vessels associated with migrating pigment epithelium cells) and area of vascular leakage that would ordinarily develop were dramatically reduced; Semi-quantitative RT-PCR analysis indicated there was upregulation of growth factors and immunohistochemistry revealed that there was an increase in neurotrophic factors within eyes of animals that received MSCs.

Conclusions/Significance

These results underscore the potential application of MSCs in treating retinal degeneration. The advantages of this non-invasive cell-based therapy are: cells are easily isolated and can be expanded in large quantity for autologous graft; hypoimmunogenic nature as allogeneic donors; less controversial in nature than other stem cells; can be readministered with minor discomfort. Therefore, MSCs may prove to be the ideal cell source for auto-cell therapy for retinal degeneration and other ocular vascular diseases.