CD133/Prom1 marks proximal mouse oviduct epithelial progenitors and adult epithelial cells with a low generative capacity.

The epithelium lining the oviduct or fallopian tube consists of multiciliated and secretory cells, which support fertilization and preimplantation development, however, its homeostasis remains poorly understood. CD133/Prom1 expression has been used as a marker to identify adult stem cell populations in various organs and often associated with cancer cells that have stem-like properties. Using an antibody targeted to CD133 and a Cre recombinase-based lineage tracing strategy, we found that CD133/Prom1 expression is not associated with a stem/progenitor population in the oviduct but marked predominantly multiciliated cells with a low generative capacity. Additionally, we have shown that CD133 is disparately localised along the oviduct during neonatal development, and that Prom1 expressing secretory cells in the ampulla rapidly transitioned to multiciliated cells and progressively migrated to the ridge of epithelial folds.

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation.

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. However, the time and cost associated with colony creation and maintenance are high. Recent advances in CRISPR-mediated genome editing have allowed the generation of somatic GEMMs (S-GEMMs) by directly targeting the cell/tissue/organ of interest. The oviduct, or fallopian tube in humans, is considered the tissue-of-origin of the most common ovarian cancer, high-grade serous ovarian carcinomas (HGSCs). HGSCs initiate in the region of the fallopian tube distal to the uterus, located adjacent to the ovary, but not the proximal fallopian tube. However, traditional mouse models of HGSC target the entire oviduct, and thus do not recapitulate the human condition. We present a method of DNA, RNA, or ribonucleoprotein (RNP) solution microinjection into the oviduct lumen and in vivo electroporation to target mucosal epithelial cells in restricted regions along the oviduct. There are several advantages of this method for cancer modeling, such as 1) high adaptability in targeting the area/tissue/organ and region of electroporation, 2) high flexibility in targeted cell types (cellular pliancy) when used in combination with specific promoters for Cas9 expression, 3) high flexibility in the number of electroporated cells (relatively low frequency), 4) no specific mouse line is required (immunocompetent disease modeling), 5) high flexibility in gene mutation combination, and 6) possibility of tracking electroporated cells when used in combination with a Cre reporter line. Thus, this cost-effective method recapitulates human cancer initiation.

Oviduct epithelial cells constitute two developmentally distinct lineages that are spatially separated along the distal-proximal axis.

Owing to technical advances in single-cell biology, the appreciation of cellular heterogeneity has increased, which has aided our understanding of organ function, homeostasis, and disease progression. The oviduct (also known as the fallopian tube) is the distalmost portion of the female reproductive tract. It is essential for reproduction and the proposed origin of high-grade serous ovarian carcinoma (HGSOC). In mammals, the oviduct is morphologically segmented along the ovary-uterus axis into four evolutionally conserved regions. It is unclear, however, if there is a diversification of epithelial cell characteristics between these regions. In this study, we identify transcriptionally distinct populations of secretory and multiciliated cells restricted to the distal and proximal regions of the oviduct. We demonstrate that distal and proximal populations are distinct lineages specified early in Müllerian duct development and are maintained separately. These results aid our understanding of epithelial development, homeostasis, and initiation of disease from the oviduct.

Modeling High-Grade Serous Ovarian Carcinoma Using a Combination of In Vivo Fallopian Tube Electroporation and CRISPR-Cas9-Mediated Genome Editing.

Ovarian cancer is the most lethal gynecologic cancer to date. High-grade serous ovarian carcinoma (HGSOC) accounts for most ovarian cancer cases, and it is most frequently diagnosed at advanced stages. Here, we developed a novel strategy to generate somatic ovarian cancer mouse models using a combination of in vivo electroporation and CRISPR-Cas9-mediated genome editing. Mutation of tumor suppressor genes associated with HGSOC in two different combinations (Brca1, Tp53, Pten with and without Lkb1) resulted in successfully generation of HGSOC, albeit with different latencies and pathophysiology. Implementing Cre lineage tracing in this system enabled visualization of peritoneal micrometastases in an immune-competent environment. In addition, these models displayed copy number alterations and phenotypes similar to human HGSOC. Because this strategy is flexible in selecting mutation combinations and targeting areas, it could prove highly useful for generating mouse models to advance the understanding and treatment of ovarian cancer. SIGNIFICANCE: This study unveils a new strategy to generate genetic mouse models of ovarian cancer with high flexibility in selecting mutation combinations and targeting areas.

Anatomical and cellular heterogeneity in the mouse oviduct-its potential roles in reproduction and preimplantation development†.

The oviduct/fallopian tube is a tube-like structure that extends from the uterus to the ovary. It is an essential reproductive organ that provides an environment for internal fertilization and preimplantation development. However, our knowledge of its regional and cellular heterogeneity is still limited. Here, we examined the anatomical complexity of mouse oviducts using modern imaging techniques and fluorescence reporter lines. We found that there are consistent coiling patterns and turning points in the coiled mouse oviduct that serve as reliable landmarks for luminal morphological regionalities. We also found previously unrecognized anatomical structures in the isthmus and uterotubal junction, which likely play roles in reproduction. Furthermore, we demarcated the ampulla-isthmus junction as a distinct region. Taken together, the oviduct mucosal epithelium has highly diverse structures with distinct epithelial cell populations, reflecting its complex functions in reproduction.

Loss of LKB1 leads to impaired epithelial integrity and cell extrusion in the early mouse embryo.

LKB1/PAR-4 is essential for the earliest polarization steps in Caenorhabditis elegans embryos and Drosophila oocytes. Although LKB1 (also known as STK11) is sufficient to initiate polarity in a single mammalian intestinal epithelial cell, its necessity in the formation and maintenance of mammalian epithelia remains unclear. To address this, we completely remove LKB1 from mouse embryos by generating maternal-zygotic Lkb1 mutants and find that it is dispensable for polarity and epithelia formation in the early embryo. Instead, loss of Lkb1 leads to the extrusion of cells from blastocyst epithelia that remain alive and can continue to divide. Chimeric analysis shows that Lkb1 is cell-autonomously required to prevent these extrusions. Furthermore, heterozygous loss of Cdh1 exacerbates the number of extrusions per blastocyst, suggesting that LKB1 has a role in regulating adherens junctions in order to prevent extrusion in epithelia.

Initiation of Hippo signaling is linked to polarity rather than to cell position in the pre-implantation mouse embryo.

In the mouse embryo, asymmetric divisions during the 8-16 cell division generate two cell types, polar and apolar cells, that are allocated to outer and inner positions, respectively. This outer/inner configuration is the first sign of the formation of the first two cell lineages: trophectoderm (TE) and inner cell mass (ICM). Outer polar cells become TE and give rise to the placenta, whereas inner apolar cells become ICM and give rise to the embryo proper and yolk sac. Here, we analyze the frequency of asymmetric divisions during the 8-16 cell division and assess the relationships between cell polarity, cell and nuclear position, and Hippo signaling activation, the pathway that initiates lineage-specific gene expression in 16-cell embryos. Although the frequency of asymmetric divisions varied in each embryo, we found that more than six blastomeres divided asymmetrically in most embryos. Interestingly, many apolar cells in 16-cell embryos were located at outer positions, whereas only one or two apolar cells were located at inner positions. Live imaging analysis showed that outer apolar cells were eventually internalized by surrounding polar cells. Using isolated 8-cell blastomeres, we carefully analyzed the internalization process of apolar cells and found indications of higher cortical tension in apolar cells than in polar cells. Last, we found that apolar cells activate Hippo signaling prior to taking inner positions. Our results suggest that polar and apolar cells have intrinsic differences that establish outer/inner configuration and differentially regulate Hippo signaling to activate lineage-specific gene expression programs.

An expanded population of CD34+ cells from frozen banked umbilical cord blood demonstrate tissue repair mechanisms of mesenchymal stromal cells and circulating angiogenic cells in an ischemic hind limb model.

Peripheral vascular disease affects ~20 % of the population over 50 years of age and is a complication of type 2 diabetes. Cell therapy studies revealed that cells from older or diabetic donors have a reduced capacity to induce tissue repair compared to healthy and younger cells. This fact greatly impedes the use of autologous cells for treatment. Umbilical cord blood CD34+ cells are a source of angiogenic cells but unlike bone marrow CD34+ angiogenic cells, achieving clinically significant cell numbers has been difficult without in vitro expansion. We report here that culturing CD34+/CD45+ blood cells from frozen umbilical cord blood units in a medium supplemented with FGF4, SCF and FLT3-ligand produced a population of cells that remain CD34+/CD45+ but have an increased capacity for tissue healing. The cultured CD34+ cells were compared directly to non-cultured CD34+ cells in a mouse model of ischemia. Cultured CD34+ cells demonstrated strong paracrine signaling as well as the capacity to differentiate into endothelial cells, smooth muscle and striated muscle. We observed an improvement in blood flow and a significant reduction in foot necrosis. A second study was completed to assess the safety of the cells. No adverse effects were associated with the injection of the cultured cells. Our method described here for culturing umbilical cord blood cells resulted in cells with a strong paracrine effect that induces substantial tissue repair in a murine model of hind limb ischemia and evidence of engraftment and differentiation of the cultured cells into new vasculature and muscle.

FGF4 is a limiting factor controlling the proportions of primitive endoderm and epiblast in the ICM of the mouse blastocyst.

The primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Although it has been shown that FGF signaling is necessary and sufficient for PE specification in the ICM, it is unknown what mechanisms control the PE/EPI proportion in the embryo. Because modulation of FGF signaling alone is sufficient to convert all ICM cells to either PE or EPI, a model has been proposed in which the amount of FGF in the embryo controls the PE/EPI proportion. To test this model, we reduced the amount of FGF4, the major FGF in the preimplantation embryo, using various genotypes of Fgf4 mutants. We observed a maternal contribution of Fgf4 in PE specification, but it was dispensable for development. In addition, upon treatment of Fgf4 mutant embryos with exogenous FGF4, we observed a progressive increase of PE proportions in an FGF4 dose dependent manner, regardless of embryo genotype. We conclude that the amount of FGF4 is limited and regulates PE/EPI proportions in the mouse embryo.

The effect of umbilical cord blood cells on outcomes after experimental traumatic spinal cord injury.

STUDY DESIGN: A cytokine expression profile of umbilical cord blood (UCB) derived multipotential stem cells (MPSC) was produced. We then transplanted MPSCs into a rat model of spinal cord injury (SCI) and assessed neurologic function as well as spinal cord histology. OBJECTIVE: To determine if MPSCs transplanted into a rat model of acute SCI would lead to a beneficial neurologic effect. SUMMARY OF BACKGROUND DATA: Conditioned medium from UCB contains factors that could promote healing of endogenous neural tissues. Previously, our laboratory has demonstrated that UCB hematopoietic cells can develop into MPSCs capable of differentiating into multiple cell types including oligodendrocyte-like cells. METHODS: We cultured MPSCs from UCB cells using fibroblast growth factor 4, stem cell factor and fms-like tyrosine kinase receptor-3 ligand supplemented serum-free medium. Using a cytokine antibody array, we produced a cytokines expression profile of MPSCs. We then transplanted MPSCs into an immunosuppressed rat model of SCI and assessed neurologic function weekly for 6 weeks by the Basso, Beattie, and Bresnahan locomotor test. The spinal cords were examined histologically and lesion areas quantified. RESULTS: We detected elevated levels of cytokines and growth factors with known neuroprotective, angiogenic, and anti-inflammatory effects in the MPSC conditioned media. The SCI rats treated with MPSCs showed a significant improvement in Basso, Beattie, and Bresnahan scores after 6 weeks compared with the group that received vehicle only. Immunohistochemistry revealed transplanted human cells were present in the injured spinal cord after 1 week, but were no longer present by 6 weeks. There was a trend for the lesion size in treated rats to be smaller than that of the control group. CONCLUSION: We conclude that UCB MPSCs improve neurologic function of rats with acute SCI, possibly by the release of factors that reduce secondary injury.

Neural progenitors, neurons and oligodendrocytes from human umbilical cord blood cells in a serum-free, feeder-free cell culture.

We have previously demonstrated that lineage negative cells (Lin(neg)) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone, muscle, endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin(neg) UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin, neurofilament, A2B5 and Sox2) are expressed in Lin(neg) cells cultured in FGF4, SCF, Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase, GalC, Olig2 and the late-stage marker MOSP are observed, as is the Schwann cell marker PMP22. In summary, Lin(neg) UCB cells, when appropriately cultured, are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation.

Bone marrow transplantation results in human donor blood cells acquiring and displaying mouse recipient class I MHC and CD45 antigens on their surface.

BACKGROUND: Mouse models of human disease are invaluable for determining the differentiation ability and functional capacity of stem cells. The best example is bone marrow transplants for studies of hematopoietic stem cells. For organ studies, the interpretation of the data can be difficult as transdifferentiation, cell fusion or surface antigen transfer (trogocytosis) can be misinterpreted as differentiation. These events have not been investigated in hematopoietic stem cell transplant models. METHODOLOGY/PRINCIPAL FINDINGS: In this study we investigated fusion and trogocytosis involving blood cells during bone marrow transplantation using a xenograft model. We report that using a standard SCID repopulating assay almost 100% of the human donor cells appear as hybrid blood cells containing both mouse and human surface antigens. CONCLUSION/SIGNIFICANCE: Hybrid cells are not the result of cell-cell fusion events but appear to be due to efficient surface antigen transfer, a process referred to as trogocytosis. Antigen transfer appears to be non-random and includes all donor cells regardless of sub-type. We also demonstrate that irradiation preconditioning enhances the frequency of hybrid cells and that trogocytosis is evident in non-blood cells in chimera mice.

A simplified procedure for hematopoietic stem cell amplification using a serum-free, feeder cell-free culture system.

Umbilical cord blood (UCB) is increasingly being used as a donor source of hematopoietic stem cells (HSCs) to treat blood malignancies. The main limitation to the widespread use of UCB is the low number of HSCs per unit. To compensate, a strategy of in vitro stem cell amplification has been attempted in different research laboratories. The major hurdle blocking success is the creation of culture conditions that support the growth of hematopoietic stem cells without their differentiation. We have designed a simple culture system for stem and progenitor cell expansion that resulted in an increased number of hematopoietic stem cells that maintain their ability to home to the bone marrow and to permanently engraft.

Identification and analysis of in vitro cultured CD45-positive cells capable of multi-lineage differentiation.

We report on a subset of cells that co-purify with CD45-positive/Lineage minus (CD45(pos)/Lin(minus)) hematopoietic cells that are capable of in vitro differentiation into multi-potential cells including cells with neuroectoderm properties. Although these cells are CD45 positive and have properties similar to CD45-negative mesenchymal progenitor cells (MPC) derived from bone marrow (BM), they are neither hematopoietic cells nor mesenchymal cells. These CD45(pos)/Lin(minus) cells can be expanded in vitro, express the stem cell genes Oct-4 and Nanog and can be induced to differentiate into endothelial cells, osteoblasts, muscle cells and neural cells at frequencies similar to those reported for bone marrow mesenchymal cells. Long-term culture of these cells followed by transplantation into NOD/SCID mice resulted in positive bone marrow stromal cell engraftment but not hematopoietic engraftment, suggesting that despite their CD45-positive status these cells do not have the same properties as hematopoietic stem cells. Clonal cell analysis determined that the culture period caused a broadening in the differentiation potential of the starting population.

Dynamic changes in cellular and microenvironmental composition can be controlled to elicit in vitro human hematopoietic stem cell expansion.

OBJECTIVE: The absence of effective strategies for the ex vivo expansion of human hematopoietic stem cells (HSCs) limits the development of many cell-based therapies. Prior attempts to stimulate HSC expansion have focused on media supplementation using cytokines and growth factors. In these cultures, cellular and microenvironmental compositions change with time. In this study, the impact of controlling these dynamic changes on HSC output is determined. MATERIALS AND METHODS: Cord blood-derived lin(-) cells were cultured for 8 days in serum-free medium supplemented with stem cell factor, Flt3 ligand, and thrombopoietin. Functional, phenotypic, and molecular (gene and protein) analyses were used to characterize dynamic changes in cellular and microenvironmental composition. The effects of these changes and the mechanism behind their effects on HSC expansion were assessed using a selection/media exchange-based global culture manipulation (GCM) technique. RESULTS: We show that the direct secretion of negative regulators by culture-generated lin(+) cells, and the indirect stimulation of cells to secrete negative regulators by culture-conditioned media, limits in vitro HSC generation. The GCM strategy was able to abrogate these effects to produce elevated numbers of LTC-ICs (14.6-fold relative to input), migrating rapid NOD/SCID repopulating cells (12.1-fold), and long-term NOD/SCID repopulating cells (5.2-fold). CONCLUSIONS: Cellular and microenvironmental changes that occur during all in vitro HSC cultures can significantly affect HSC output through the direct or indirect secretion of negative regulators. This study provides insight into the mechanisms regulating HSC fate in vitro and describes a novel methodology to regulate overall in vitro microenvironmental dynamics to enable the generation of clinically relevant numbers of HSCs.