Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest^1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal''s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.     

Genomic Instability in Pluripotent Stem Cells: Implications for Clinical Applications

Human pluripotent stem cells (hPSCs) are known to acquire genomic changes as they proliferate and differentiate. Despite concerns that these changes will compromise the safety of hPSC-derived cell therapy, there is currently scant evidence linking the known hPSC genomic abnormalities with malignancy. For the successful use of hPSCs for clinical applications, we will need to learn to distinguish between innocuous genomic aberrations and those that may cause tumors. To minimize any effects of acquired mutations on cell therapy, we strongly recommend that cells destined for transplant be monitored throughout their preparation using a high-resolution method such as SNP genotyping.     

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies¹. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia^2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma^2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds⁹. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division¹⁰. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity². This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry². The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.     

Allogeneic Human Mesenchymal Stem Cells Restore Epithelial Protein Permeability in Cultured Human Alveolar Type II Cells by Secretion of Angiopoietin-1

Acute lung injury is characterized by injury to the lung epithelium that leads to impaired resolution of pulmonary edema and also facilitates accumulation of protein-rich edema fluid and inflammatory cells in the distal airspaces of the lung. Recent in vivo and in vitro studies suggest that mesenchymal stem cells (MSC) may have therapeutic value for the treatment of acute lung injury. Here we tested the ability of human allogeneic mesenchymal stem cells to restore epithelial permeability to protein across primary cultures of polarized human alveolar epithelial type II cells after an inflammatory insult. Alveolar epithelial type II cells were grown on a Transwell plate with an air-liquid interface and injured by cytomix, a combination of IL-1β, TNFα, and IFNγ. Protein permeability measured by ¹³¹I-labeled albumin flux was increased by 5-fold over 24 h after cytokine-induced injury. Co-culture of human MSC restored type II cell epithelial permeability to protein to control levels. Using siRNA knockdown of potential paracrine soluble factors, we found that angiopoietin-1 secretion was responsible for this beneficial effect in part by preventing actin stress fiber formation and claudin 18 disorganization through suppression of NFκB activity. This study provides novel evidence for a beneficial effect of MSC on alveolar epithelial permeability to protein.     

High Oxygen Condition Facilitates the Differentiation of Mouse and Human Pluripotent Stem Cells into Pancreatic Progenitors and Insulin-producing Cells

Pluripotent stem cells have potential applications in regenerative medicine for diabetes. Differentiation of stem cells into insulin-producing cells has been achieved using various protocols. However, both the efficiency of the method and potency of differentiated cells are insufficient. Oxygen tension, the partial pressure of oxygen, has been shown to regulate the embryonic development of several organs, including pancreatic β-cells. In this study, we tried to establish an effective method for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells by culturing under high oxygen (O₂) conditions. Treatment with a high O₂ condition in the early stage of differentiation increased insulin-positive cells at the terminus of differentiation. We found that a high O₂ condition repressed Notch-dependent gene Hes1 expression and increased Ngn3 expression at the stage of pancreatic progenitors. This effect was caused by inhibition of hypoxia-inducible factor-1α protein level. Moreover, a high O₂ condition activated Wnt signaling. Optimal stage-specific treatment with a high O₂ condition resulted in a significant increase in insulin production in both mouse embryonic stem cells and human iPSCs and yielded populations containing up to 10% C-peptide-positive cells in human iPSCs. These results suggest that culturing in a high O₂ condition at a specific stage is useful for the efficient generation of insulin-producing cells.     

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.     

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.     

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.     

Induction of Cancerous Stem Cells during Embryonic Stem Cell Differentiation

Stem cell maintenance depends on their surrounding microenvironment, and aberrancies in the environment have been associated with tumorigenesis. However, it remains to be elucidated whether an environmental aberrancy can act as a carcinogenic stress for cellular transformation of differentiating stem cells into cancer stem cells. Here, utilizing mouse embryonic stem cells as a model, it was illustrated that environmental aberrancy during differentiation leads to the emergence of pluripotent cells showing cancerous characteristics. Analogous to precancerous stages, DNA lesions were spontaneously accumulated during embryonic stem cell differentiation under aberrational environments, which activates barrier responses such as senescence and apoptosis. However, overwhelming such barrier responses, piled-up spheres were subsequently induced from the previously senescent cells. The sphere cells exhibit aneuploidy and dysfunction of the Arf-p53 module as well as enhanced tumorigenicity and a strong self-renewal capacity, suggesting development of cancerous stem cells. Our current study suggests that stem cells differentiating in an aberrational environment are at risk of cellular transformation into malignant counterparts.     

Effect of Thrombin on Human Amnion Mesenchymal Cells,Mouse Fetal Membranes,and Preterm Birth

Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE₂ in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE₂ content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion.     

牛乳腺上皮细胞的分离培养及其生物学特性

采用胶原酶消化法和胰蛋白酶选择性消化法分离、培养和纯化牛乳腺上皮细胞。形态学观察表明,培养的细胞具有典型的上皮细胞形态特征;染色体分析结果表明,培养的细胞具有正常的染色体数目。通过荧光免疫细胞染色方法鉴定了培养的细胞表达上皮细胞特异的角蛋白5和8。该细胞在添加胰岛素、氢化可的松以及羊催乳素的无血清培养液中诱导培养时,用RT-PCR方法检测到了β-酪蛋白基因的转录。这些结果表明,分离培养的细胞是乳腺上皮细胞,这些细胞在诱导培养的条件下能够转录表达β-酪蛋白。     

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.     

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in ¹. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases ². The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands ³. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways ³.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.     

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 10⁶ acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.     

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c⁺), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45^hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.     

SD大鼠骨髓间充质干细胞体外原代培养及鉴定

目的：探讨骨髓间充质干细胞（BMSCs）体外分离培养以及扩增的方法并鉴定。方法：取100g左右雄性SD大鼠后肢股骨、胫骨骨髓,原代全骨髓培养法,多次传代纯化,体外扩增后,观察细胞形态,并免疫组化及流式细胞仪检测cd34、cd90、cd105细胞因子,鉴定是否为BMSCs。结果：所获取的细胞呈长梭形,呈现特征性的漩涡状生长,CD34阴性,CD90、CD105阳性。结论：利用全骨髓培养法成功分离骨髓间充质干细胞,10代以内的细胞纯度高,活性好。全骨髓培养较为简便、易行。     

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 ¹ and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 ². Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 μm pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.     

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5^cre:ZsGreen^l/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.