scholarly journals OR31-03 Single-Cell Profiling of Adult Human Ovarian Cortex Reveals Six Main Cell Types but No Germline Stem Cells

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Magdalena Wagner ◽  
Masahito Yoshihara ◽  
Iyadh Douagi ◽  
Anastasios Damdimopoulos ◽  
Sarita Panula ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Cell Surface ◽  
Single Cell ◽  
Surface Antigen ◽  
Cell Types ◽  
Cell Surface Antigen ◽  
Germline Stem Cells ◽  
Ovarian Cortex ◽  
Main Cell

Abstract The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been suggested that oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Our study aimed at comprehensive characterization of all cell types present in the ovarian cortex, including the previously reported oogonial stem cells. We developed methods to dissociate human ovarian cortex to a viable single cell solution allowing subsequent analysis by single cell transcriptomic profiling and cell surface antigen screening. In all analyses, cells captured by DDX4 antibodies (DDX4 Ab+) were included as a reference. High quality ovarian cortex tissue from gender reassignement and caesarean section patients was used in the analyses. Our single cell transcriptomic analyses based on >24,000 cells revealed the presence of six main cell types in ovarian cortex; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Surface marker screening showed robust expression of 43 cell surface antigens in ovarian cortex cells. With the help of transcriptomic and cell surface antigen profiles, the DDX4 Ab+ cells were identified as perivascular cells. This finding was validated by immunostaining of ovarian tissue showing DDX4 Ab+ cells lining CD31 positive endothelial cells of blood vessels. To search for germline stem cells on a broader front, we compared our data with human fetal ovary cells including pre-meiotic germ cells (Li et al. 2017) and found no evidence for the presence of germ line stem cells of any kind in adult human ovarian cortex. In summary, we provide the first cell map of human ovarian cortex. Our results demonstrate six main cell types, but cannot provide support to the existence of oogonial stem cells. This dataset will be a valuable tool for studying the role of specific cell populations in ovarian biology, dissecting causes of infertility, and developing novel assisted reproductive technologies or even contraceptives.

scholarly journals Single cell map of the human ovarian cortex

2019 ◽  
Author(s):  
Magdalena Wagner ◽  
Masahito Yoshihara ◽  
Iyadh Douagi ◽  
Anastasios Damdimopoulos ◽  
Sarita Panula ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Ovarian Reserve ◽  
Structural Changes ◽  
Cell Types ◽  
Germline Stem Cells ◽  
Perivascular Cells ◽  
Hormone Production ◽  
Ovarian Cortex ◽  
Outer Lining

ABSTRACTThe human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. We analysed on a single cell level the transcriptome and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our single cell mapping reveals transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data does not support the existence of germline stem cells in adult human ovaries thereby reinforcing the dogma of a limited ovarian reserve.

scholarly journals Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Magdalena Wagner ◽  
Masahito Yoshihara ◽  
Iyadh Douagi ◽  
Anastasios Damdimopoulos ◽  
Sarita Panula ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Ovarian Reserve ◽  
Structural Changes ◽  
Single Cell Analysis ◽  
Cell Types ◽  
Germline Stem Cells ◽  
Perivascular Cells ◽  
Hormone Production ◽  
Ovarian Cortex

AbstractThe human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Here, we report single-cell transcriptomes and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our data identify transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data do not support the existence of germline stem cells in adult human ovaries, thereby reinforcing the dogma of a limited ovarian reserve.

scholarly journals Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells

2017 ◽  
Vol 26 (6) ◽  
pp. 1089-1102 ◽  
Author(s):  
Kunikazu Tsuji ◽  
Miyoko Ojima ◽  
Koji Otabe ◽  
Masafumi Horie ◽  
Hideyuki Koga ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Surface ◽  
Surface Antigen ◽  
Antigen Expression ◽  
Cell Surface Antigen ◽  
Surface Antigen Expression

Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency

2014 ◽  
Vol 306 (12) ◽  
pp. C1108-C1118 ◽  
Author(s):  
Steven C. Tsai ◽  
David F. Chang ◽  
Chang-Mu Hong ◽  
Ping Xia ◽  
Dinithi Senadheera ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Surface ◽  
Surface Antigen ◽  
Embryonic Stem ◽  
Antigen Expression ◽  
Cell Surface Antigen ◽  
Cloning Efficiency ◽  
Self Renewal ◽  
Surface Antigen Expression

Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.

In vitro Three Dimensional Scaffold-free Construct of Human Adipose-derived Stem Cells in Coculture with Endothelial Cells and Fibroblasts

2017 ◽  
Vol 68 (6) ◽  
pp. 1341-1344
Author(s):  
Grigore Berea ◽  
Gheorghe Gh. Balan ◽  
Vasile Sandru ◽  
Paul Dan Sirbu
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Endothelial Cells ◽  
Single Cell ◽  
Cell Line ◽  
Bone Repair ◽  
Umbilical Vein ◽  
Human Fibroblasts ◽  
Three Dimensional ◽  
Adipose Derived Stem Cells

Complex interactions between stem cells, vascular cells and fibroblasts represent the substrate of building microenvironment-embedded 3D structures that can be grafted or added to bone substitute scaffolds in tissue engineering or clinical bone repair. Human Adipose-derived Stem Cells (hASCs), human umbilical vein endothelial cells (HUVECs) and normal dermal human fibroblasts (NDHF) can be mixed together in three dimensional scaffold free constructs and their behaviour will emphasize their potential use as seeding points in bone tissue engineering. Various combinations of the aforementioned cell lines were compared to single cell line culture in terms of size, viability and cell proliferation. At 5 weeks, viability dropped for single cell line spheroids while addition of NDHF to hASC maintained the viability at the same level at 5 weeks Fibroblasts addition to the 3D construct of stem cells and endothelial cells improves viability and reduces proliferation as a marker of cell differentiation toward osteogenic line.

Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

1979 ◽  
Author(s):  
S. Korach ◽  
D. Ngo
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Vascular Endothelial Cells ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Endothelial Damage ◽  
Antibody Concentration ◽  
High Yields ◽  
Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

scholarly journals Adult tissue-resident stem cells—fact or fiction?

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Deepa Bhartiya
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Adult Stem Cells ◽  
Cell Types ◽  
Specific Cell ◽  
Tissue Specific ◽  
Cardiac Tissues ◽  
Adult Tissues ◽  
Resident Stem Cells ◽  
Abundant Cytoplasm

AbstractLife-long tissue homeostasis of adult tissues is supposedly maintained by the resident stem cells. These stem cells are quiescent in nature and rarely divide to self-renew and give rise to tissue-specific “progenitors” (lineage-restricted and tissue-committed) which divide rapidly and differentiate into tissue-specific cell types. However, it has proved difficult to isolate these quiescent stem cells as a physical entity. Recent single-cell RNAseq studies on several adult tissues including ovary, prostate, and cardiac tissues have not been able to detect stem cells. Thus, it has been postulated that adult cells dedifferentiate to stem-like state to ensure regeneration and can be defined as cells capable to replace lost cells through mitosis. This idea challenges basic paradigm of development biology regarding plasticity that a cell enters point of no return once it initiates differentiation. The underlying reason for this dilemma is that we are putting stem cells and somatic cells together while processing for various studies. Stem cells and adult mature cell types are distinct entities; stem cells are quiescent, small in size, and with minimal organelles whereas the mature cells are metabolically active and have multiple organelles lying in abundant cytoplasm. As a result, they do not pellet down together when centrifuged at 100–350g. At this speed, mature cells get collected but stem cells remain buoyant and can be pelleted by centrifuging at 1000g. Thus, inability to detect stem cells in recently published single-cell RNAseq studies is because the stem cells were unknowingly discarded while processing and were never subjected to RNAseq. This needs to be kept in mind before proposing to redefine adult stem cells.

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