scholarly journals Ovarian Decellularized Bioscaffolds Provide an Optimal Microenvironment for Cell Growth and Differentiation In Vitro

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2126
Author(s):  
Georgia Pennarossa ◽  
Teresina De Iorio ◽  
Fulvio Gandolfi ◽  
Tiziana A. L. Brevini
Keyword(s):  
Ex Vivo ◽  
Fertility Restoration ◽  
Dermal Fibroblasts ◽  
Decellularized Extracellular Matrix ◽  
Ovarian Cells ◽  
Novel Approach ◽  
Definitive Solution ◽  
Cell Growth And Differentiation ◽  
Ex Vivo Culture

Ovarian failure is the most common cause of infertility. Although numerous strategies have been proposed, a definitive solution for recovering ovarian functions and restoring fertility is currently unavailable. One innovative alternative may be represented by the development of an “artificial ovary” that could be transplanted in patients for re-establishing reproductive activities. Here, we describe a novel approach for successful repopulation of decellularized ovarian bioscaffolds in vitro. Porcine whole ovaries were subjected to a decellularization protocol that removed the cell compartment, while maintaining the macrostructure and microstructure of the original tissue. The obtained bioscaffolds were then repopulated with porcine ovarian cells or with epigenetically erased porcine and human dermal fibroblasts. The results obtained demonstrated that the decellularized extracellular matrix (ECM)-based scaffold may constitute a suitable niche for ex vivo culture of ovarian cells. Furthermore, it was able to properly drive epigenetically erased cell differentiation, fate, and viability. Overall, the method described represents a powerful tool for the in vitro creation of a bioengineered ovary that may constitute a promising solution for hormone and fertility restoration. In addition, it allows for the creation of a suitable 3D platform with useful applications both in toxicological and transplantation studies.

Assessment of Long-Term in vitro Multiplied Human Wharton’s Jelly-Derived Mesenchymal Stem Cells prior to Their Use in Clinical Administration

2021 ◽  
pp. 1-11
Author(s):  
Urvi Panwar ◽  
Kanchan Mishra ◽  
Parizad Patel ◽  
Sumit Bharadva ◽  
Salil Vaniawala ◽  
...  
Keyword(s):  
Telomere Length ◽  
Replicative Senescence ◽  
Ex Vivo ◽  
Wharton’S Jelly ◽  
Cellular Aging ◽  
Human Umbilical Cord ◽  
Surface Markers ◽  
Wharton's Jelly ◽  
Ex Vivo Culture

The quantity of mesenchymal stem/stromal cells (MSCs) required for a particular therapy demands their subsequent expansion through ex vivo culture. During in vitro multiplication, they undergo replicative senescence which may alter their genetic stability. Therefore, this study was aimed to analyze cellular, molecular, and chromosomal alterations in Wharton’s jelly-derived MSCs (WJ-MSCs) during their in vitro sequential passages, where WJ-MSCs were sequentially passaged up to P14 and cells were evaluated at an interval of P2, P6, P10, and P14. They were examined for their morphology, tumorigenicity, surface markers, stemness markers, DNA damage, chromosomal aberration, and telomere length. We have processed five full-term delivered human umbilical cord samples to obtain WJ-MSCs. Morphological appearance observed at initial stages was small fine spindle-shaped WJ-MSCs which were transformed to flat, long, and broader cells in later passages. The cell proliferation rate was gradually decreased after the 10th passage. WJ-MSCs have expressed stemness markers OCT-4 and NANOG, while they showed high expression of positive surface markers CD90 and CD105 and lower expression of CD34 and CD45. They were non-tumorigenic with slow cellular aging during subsequent passages. There was no chromosomal abnormality up to the 14th passage, while increase in comet score and decrease in telomere length were observed in later passages. Hence, our study suggests that early and middle passaged (less than P10) WJ-MSCs are good candidates for clinical administration for treatment.

scholarly journals Single-Cell Receptor Quantification of an In Vitro Coculture Angiogenesis Model Reveals VEGFR, NRP1, Tie2, and PDGFR Regulation and Endothelial Heterogeneity

Processes ◽  
2019 ◽  
Vol 7 (6) ◽  
pp. 356 ◽  
Author(s):  
Si Chen ◽  
P. I. Imoukhuede
Keyword(s):  
Systems Biology ◽  
Growth Factor ◽  
Plasma Membranes ◽  
Temporal Dynamics ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Dermal Fibroblasts ◽  
Steady Increase

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for both normal development and numerous pathologies. Systems biology has offered a unique approach to study angiogenesis by profiling tyrosine kinase receptors (RTKs) that regulate angiogenic processes and computationally modeling RTK signaling pathways. Historically, this systems biology approach has been applied on ex vivo angiogenesis assays, however, these assays are difficult to quantify and limited in their potential of temporal analysis. In this study, we adopted a simple two-dimensional angiogenesis assay comprised of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) and examined temporal dynamics of a panel of six RTKs and cell heterogeneity up to 17 days. We observed ~2700 VEGFR1 (vascular endothelial growth factor receptor 1) per cell on 24-h-old cocultured HDF plasma membranes, which do not express VEGFR when cultured alone. We observed 4000–8100 VEGFR2 per cell on cocultured HUVEC plasma membranes throughout endothelial tube formation. We showed steady increase of platelet-derived growth factor receptors (PDGFRs) on cocultured HDF plasma membranes, and more interestingly, 1900–2900 PDGFRβ per plasma membrane were found on HUVECs within the first six hours of coculturing. These quantitative findings will offer us insights into molecular regulation during angiogenesis and help assess in vitro tube formation models and their physiological relevance.

scholarly journals Differential Maintenance of Primitive Human SCID-Repopulating Cells, Clonogenic Progenitors, and Long-Term Culture-Initiating Cells After Incubation on Human Bone Marrow Stromal Cells

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 641-650 ◽  
Author(s):  
Olga I. Gan ◽  
Barbara Murdoch ◽  
Andre Larochelle ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Stromal Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Long Term Culture ◽  
Cell Engraftment ◽  
Ex Vivo Culture

Abstract Many experimental and clinical protocols are being developed that involve ex vivo culture of human hematopoietic cells on stroma or in the presence of cytokines. However, the effect of these manipulations on primitive hematopoietic cells is not known. Our severe combined immune-deficient mouse (SCID)-repopulating cell (SRC) assay detects primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of immune-deficient non-obese diabetic/SCID (NOD/SCID) mice. We have examined here the maintenance of SRC, colony-forming cells (CFC), and long-term culture-initiating cells (LTC-IC) during coculture of adult human BM or umbilical cord blood (CB) cells with allogeneic human stroma. Transplantation of cultured cells in equivalent doses as fresh cells resulted in lower levels of human cell engraftment after 1 and 2 weeks of culture for BM and CB, respectively. Similar results were obtained using CD34+-enriched CB cells. By limiting dilution analysis, the frequency of SRC in BM declined sixfold after 1 week of culture. In contrast to the loss of SRC as measured by reduced repopulating capacity, the transplanted inocula of cultured cells frequently contained equal or higher numbers of CFC and LTC-IC compared with the inocula of fresh cells. The differential maintenance of CFC/LTC-IC and SRC suggests that SRC are biologically distinct from the majority of these in vitro progenitors. This report demonstrates the importance of the SRC assay in the development of ex vivo conditions that will allow maintenance of primitive human hematopoietic cells with repopulating capacity.

scholarly journals Quantitative Analysis Reveals Expansion of Human Hematopoietic Repopulating Cells After Short-term Ex Vivo Culture

1997 ◽  
Vol 186 (4) ◽  
pp. 619-624 ◽  
Author(s):  
Mickie Bhatia ◽  
Dominique Bonnet ◽  
Ursula Kapp ◽  
Jean C.Y. Wang ◽  
Barbara Murdoch ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Fold Increase ◽  
Culture Conditions ◽  
Nonobese Diabetic ◽  
Crucial Component ◽  
Cord Blood Cells ◽  
Ex Vivo Culture

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays, knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38− cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38− cells and colony-forming cells, respectively, as well as a 2- to 4-fold increase in SRC after 4 d of culture. However, after 9 d of culture, all SRC were lost, despite further increases in total cells, CFC content, and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation, and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells.

Cytokine Treatment of CD34+ Cord Blood Cells with G-CSF, GM-CSF, or SCF During 48 Hours of Ex Vivo Culture Alters CD26/DPPIV Peptidase Activity and Subsequent Engraftment Into NSG Immunodeficient Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1460-1460
Author(s):  
Laura A Paganessi ◽  
Lydia Luy Tan ◽  
Sucheta Jagan ◽  
Robin Frank ◽  
Antonio M. Jimenez ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Gm Csf ◽  
Cytokine Treatment ◽  
Ex Vivo Culture ◽  
Inhibitor Treatment

Abstract Abstract 1460 Many patients with hematologic malignancies choose hematopoietic stem cell transplantation (HSCT) as a treatment option. The most common source of Hematopoietic Stem and Progenitor Cells (HSC/HPC) for adult recipients is mobilized Peripheral Blood (mobPB). Limited quantities of HSC/HPC obtainable from an umbilical cord restricts its use for adult recipients. Ex vivo treatment of umbilical cord blood (CB) with cytokines and growth factors is being used to expand the population of cord blood HSC/HPCs in hopes of obtaining higher numbers of transplantable CB cells. In addition, cytokines and growth factors are often utilized post-transplant in an attempt to improve the rate of immune reconstitution. It has been previously reported that granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage-colony-stimulating factor (GM-CSF) up-regulate CD26 (dipeptidyl peptidase IV/DPPIV) activity on freshly isolated CD34+ CB cells within 18 hours of culture [Christopherson, et al. Exp Hematol 2006]. Separate studies have demonstrated that treatment of uncultured CD34+ CB cells with the CD26 inhibitor Diprotin A increases transplant efficiency into immunodeficient mice [Christopherson, et al. Stem Cells Dev. 2007]. We evaluated here the in vitro and in vivo effects of CD26 inhibitor treatment on previously frozen CB CD34+ cells cultured ex vivo with G-CSF, GM-CSF or SCF for 48 hours. We examined CD26 expression by multivariate flow cytometry, CD26 activity using the established chromogenic CD26 substrate, Gly-Pro-p-nitroanilide (Gly-Pro-pNA), and SDF-1α induced migration and adhesion. In vivo, we examined long-term engraftment in NSG (NOD/SCID/IL2Rγnull) immunodeficient mice. After 48 hours of culture with cytokine treatment we observed altered CD26 expression on CD34+ CB cells. There was both an increase in the percentage of CD26+ cells and the mean fluorescence intensity (MFI) of CD26. Additionally, CD26 activity was 1.20, 1.59, 1.58, and 1.65 fold greater after ex vivo culture in untreated, G-CSF, GM-CSF and SCF treated CB CD34+ cells respectively compared to the CD26 activity prior to culture. The increase in CD26 activity as a result of treatment with G-CSF (p≤ 0.01), GM-CSF (p≤ 0.05) or SCF (p≤ 0.01) was significantly higher than the CD26 activity measured in the untreated cells following 48 hours of culture. Post-culture treatment with the CD26 inhibitor, Diprotin A, significantly improved SDF-1α induced migration and adhesion of cultured CD34+ CB cells in vitro, particularly in G-CSF treated cells (p≤ 0.05). Diprotin A treatment of CD34+ CB cells previously treated with G-CSF also significantly increased the long-term in vivo engraftment of stem and progenitor (CD34+CD38-, p=0.032), monocyte (CD14+, p=0.015), and megakaryocyte/platelet (CD61+, p=0.020) cells in the bone marrow of NSG mice. CD26 has been previously shown to cleave SDF-1 (stromal cell-derived factor 1/CXCL12). After cleavage, SDF-1 retains its ability to bind to its receptor (CXCR4) but no longer signals. SDF-1 is a powerful chemoattractant and has been shown to be important in mobilization, homing, and engraftment of HSCs and HPCs. This study demonstrates the influence of ex vivo culture and the effect of cytokine treatment on CD26 activity and subsequent biologic function related to HSCT. All three cytokines studied caused a significant increase in enzymatic activity at 48 hours compared to untreated cells. The up-regulation of CD26 protein expression caused by cytokine treatment for 48 hours, in particular G-CSF, had a significant impact on SDF-1 stimulated migration and adhesion. This was demonstrated in vitro by the improvement in cell function after CD26 inhibitor treatment and in vivo by the improved engraftment seen in the G-CSF treated cells with CD26 inhibitor treatment. These experiments suggest that combining CD26 inhibitor treatment following culture with G-CSF treatment during culture has the greatest overall benefit in engraftment outcome. By increasing our understanding of the effects of exogenous cytokines during culture on trafficking, ex vivo expanded CB has the potential to become a more effective means of not only increasing numbers of CB HSC/HPCs but also engraftment outcomes. This would ultimately allow expanded cord blood to become a more viable option for HSCT. Disclosures: No relevant conflicts of interest to declare.

Comparison of in vivo skin and in vitro blood lymphocyte models for the prediction of late normal tissue responses in breast radiotherapy (RT) patients.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 11116-11116
Author(s):  
Melvin Chua ◽  
Navita Somaiah ◽  
Sue Davies ◽  
Lone Gothard ◽  
Kai Rothkamm ◽  
...  
Keyword(s):  
Late Effects ◽  
Normal Tissue ◽  
Ex Vivo ◽  
Predictive Marker ◽  
Dermal Fibroblasts ◽  
Clinical Severity ◽  
Breast Size ◽  
Dsb Repair

11116 Background: Critical opinions for the lack of success of DNA double-strand break (DSB) repair as a predictive marker of normal tissue radiosensitivity include the argument that in vitro cellular responses correlate poorly with in vivo responses due to the modifying influence of tissue environment. In this study, we test the hypothesis that a DNA damage assay based on in vivo irradiated skin tissues better predicts clinical responses in human skin, as opposed to the same assay performed in ex vivo irradiated lymphocytes. Methods: DSB levels (24 h post-4 Gy) were quantified using γH2AX/53BP1 immunostaining in irradiated skin tissues and G0 lymphocytes of 35 breast RT patients. Patients were selected on the basis of late RT effects in their breast and individuals with marked or minimal effects were classified as cases and controls, respectively. Risk factors of late effects established from multivariate analyses of outcomes of two breast RT trials were also considered in patient selection. They were 1) total RT dose, 2) RT dosimetry, 3) tumour bed boost, 4) breast size, 5) surgical cavity, and 6) axillary treatment. Results: Clinical parameters were balanced in both patient groups. Residual foci levels in skin epidermis and dermis were comparable between cases (n = 20) and controls (n = 15). Mean foci per cell were 3.29 in cases, 2.80 in controls for dermal fibroblasts (p = 0.07); 3.28 in cases, 2.60 in controls for endothelial cells (p = 0.08); 2.87 in cases, 2.41 in controls for superficial keratinocytes (p = 0.45); 2.32 in cases, 2.35 in controls for basal keratinocytes (p = 0.27). Residual foci levels in lymphocytes were however significantly higher among cases (foci per cell = 12.1) compared to controls (foci per cell = 10.3, p = 0.01). Of the different cell types, only residual foci levels of dermal fibroblasts and lymphocytes correlated with clinical severity (R = 0.722, p < 0.001; 0.593, p = 0.01, respectively). Interestingly, foci levels were not correlated between skin cells and lymphocytes of the same patients. Conclusions: DSB repair of ex vivo irradiated lymphocytes appears to be a better predictive marker of late effects to breast RT than DSB repair of in vivo irradiated skin.

scholarly journals Functional Heterogeneity of Human CD34+ Cells Isolated in Subcompartments of the G0 /G1 Phase of the Cell Cycle

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4384-4393 ◽  
Author(s):  
André Gothot ◽  
Robert Pyatt ◽  
Jon McMahel ◽  
Susan Rice ◽  
Edward F. Srour
Keyword(s):  
Cell Cycle ◽  
Ex Vivo ◽  
G1 Phase ◽  
Dna And Rna ◽  
Human Cd34 ◽  
Rna Content ◽  
Cd34 Cells ◽  
Ex Vivo Culture

Using simultaneous Hoechst 33342 (Hst) and Pyronin Y (PY) staining for determination of DNA and RNA content, respectively, human CD34+ cells were isolated in subcompartments of the G0 /G1 phase of the cell cycle by flow cytometric cell sorting. In both bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells, primitive long-term hematopoietic culture-initiating cell (LTHC-IC) activity was higher in CD34+ cells isolated in G0 (G0CD34+ cells) than in those residing in G1 (G1CD34+ cells). However, as MPB CD34+ cells displayed a more homogeneous cell-cycle status within the G0 /G1 phase and a relative absence of cells in late G1 , DNA/RNA fractionation was less effective in segregating LTHC-IC in MPB than in BM. BM CD34+ cells belonging to four subcompartments of increasing RNA content within the G0 /G1 phase were evaluated in functional assays. The persistence of CD34 expression in suspension culture was inversely correlated with the initial RNA content of test cells. Multipotential progenitors were present in G0 or early G1 subcompartments, while lineage-restricted granulomonocytic progenitors were more abundant in late G1 . In vitro hematopoiesis was maintained for up to 6 weeks with G0CD34+ cells, whereas production of clonogenic progenitors was more limited in cultures initiated with G1CD34+ cells. To test the hypothesis that primitive LTHC-ICs would reenter a state of relative quiescence after in vitro division, BM CD34+ cells proliferating in ex vivo cultures were identified from their quiescent counterparts by a relative loss of membrane intercalating dye PKH2, and were further fractionated with Hst and PY. The same functional hierarchy was documented within the PKH2dim population whereby LTHC-IC frequency was higher for CD34+ cells reselected in G0 after in vitro division than for CD34+ cells reisolated in G1 or in S/G2 + M. However, the highest LTHC-IC frequency was found in quiescent PKH2bright CD34+ cells. Together, these results support the concept that cells with distinct hematopoietic capabilities follow different pathways during the G0 /G1 phase of the cell cycle both in vivo and during ex vivo culture.

scholarly journals Novel Rotational Combination Regimen of Skin Topicals Improves Facial Photoaging: Efficacy Demonstrated in Double-Blinded Clinical Trials and Laboratory Validation

2021 ◽  
Vol 8 ◽  
Author(s):  
Lisa DiNatale ◽  
Jolanta Idkowiak-Baldys ◽  
Young Zhuang ◽  
Anthony Gonzalez ◽  
Thomas J. Stephens ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Ex Vivo ◽  
Dermal Fibroblasts ◽  
Combination Regimen ◽  
Crow’S Feet ◽  
Skin Explants ◽  
Study Methodology ◽  
Double Blinded

Topical antiaging products are often a first-line intervention to counter visible signs of facial photoaging, aiming for sustained cosmetic improvement. However, prolonged application of a single active topical compound was observed clinically to lead to a plateau effect in improving facial photoaging. In view of this, we set out to reduce this effect systematically using a multi-tiered approach with laboratory evidence and clinical trials. The objective of the study was to evaluate the effects of active topical ingredients applied either alone, in combination, or in a rotational manner on modulation of facial photoaging. The study methodology included in vitro, organotypic, and ex vivo skin explants; in vivo biopsy study; as well as clinical trials. We demonstrate for the first time that a pair of known antiaging ingredients applied rotationally, on human dermal fibroblasts, maximized pro-collagen I production. Indeed, rotational treatment with retinol and phytol/glycolic acid (PGA) resulted in better efficacy than application of each active ingredient alone as shown by explants and in vivo biopsy study, with penetration of active ingredients confirmed by Raman spectroscopy. Furthermore, two split-face, randomized, double-blinded clinical trials were conducted, one for 12 months to compare treated vs. untreated and the other for 6 months followed by a 2-month regression to compare treated vs. commercially marketed products. In both studies, rotational regimen showed superior results to its matching comparison as assessed by clinical grading and image analysis of crow's feet wrinkles. In conclusion, rotational regimen using retinol and PGA is effective in treating facial photoaging signs with long-lasting benefits.

scholarly journals Cell-autonomous FLT3L shedding via ADAM10 mediates conventional dendritic cell development in mouse spleen

2019 ◽  
Vol 116 (29) ◽  
pp. 14714-14723 ◽  
Author(s):  
Kohei Fujita ◽  
Svetoslav Chakarov ◽  
Tetsuro Kobayashi ◽  
Keiko Sakamoto ◽  
Benjamin Voisin ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Embryonic Fibroblasts ◽  
Major Pathway ◽  
Peripheral Tissues ◽  
A Cell ◽  
Membrane Bound ◽  
A Disintegrin And Metalloproteinase ◽  
Ex Vivo Culture ◽  
Culture Supernatants

Conventional dendritic cells (cDCs) derive from bone marrow (BM) precursors that undergo cascades of developmental programs to terminally differentiate in peripheral tissues. Pre-cDC1s and pre-cDC2s commit in the BM to each differentiate into CD8α+/CD103+ cDC1s and CD11b+ cDC2s, respectively. Although both cDCs rely on the cytokine FLT3L during development, mechanisms that ensure cDC accessibility to FLT3L have yet to be elucidated. Here, we generated mice that lacked a disintegrin and metalloproteinase (ADAM) 10 in DCs (Itgax-cre × Adam10-fl/fl; ADAM10∆DC) and found that ADAM10 deletion markedly impacted splenic cDC2 development. Pre-cDC2s accumulated in the spleen with transcriptomic alterations that reflected their inability to differentiate and exhibited abrupt failure to survive as terminally differentiated cDC2s. Induced ADAM10 ablation also led to the reduction of terminally differentiated cDC2s, and restoration of Notch signaling, a major pathway downstream of ADAM10, only modestly rescued them. ADAM10∆DC BM failed to generate cDC2s in BM chimeric mice with or without cotransferred ADAM10-sufficient BM, indicating that cDC2 development required cell-autonomous ADAM10. We determined cDC2s to be sources of soluble FLT3L, as supported by decreased serum FLT3L concentration and the retention of membrane-bound FLT3L on cDC2 surfaces in ADAM10∆DC mice, and by demonstrating the release of soluble FLT3L by cDC2 in ex vivo culture supernatants. Through in vitro studies utilizing murine embryonic fibroblasts, we determined FLT3L to be a substrate for ADAM10. These data collectively reveal cDC2s as FLT3L sources and highlight a cell-autonomous mechanism that may enhance FLT3L accessibility for cDC2 development and survival.

Sign in / Sign up

Export Citation Format

Share Document