In vitro growth of immature bovine follicles and oocytes

2020 ◽  
Vol 32 (2) ◽  
pp. 1 ◽  
Author(s):  
Evelyn E. Telfer ◽  
Kenichiro Sakaguchi ◽  
Yvonne L. Clarkson ◽  
Marie McLaughlin
Keyword(s):  
Stem Cell ◽  
Assisted Reproduction ◽  
Culture System ◽  
Developmental Stages ◽  
In Vitro Growth ◽  
Primordial Follicles ◽  
Domestic Species ◽  
Stem Cell Derivation ◽  
Major Impediment

The limitation in the supply of mature, fertilisable oocytes constitutes a major impediment to increasing the success of assisted reproduction, stem cell derivation and cloning in domestic species. Techniques are being developed to grow immature oocytes invitro that have the potential to increase the supply of oocytes. Mouse oocytes can be cultured from initial stages of development to maturity, and live young have been produced, but for domestic species, such as cows, with long growth periods, invitro systems that allow complete growth of oocytes contained within primordial follicles to maturity is technically challenging and has not yet been achieved. For cows, several culture systems have been developed that support specific developmental stages, but a multistep culture system will be required for complete growth invitro. This review highlights the steps that will be required to achieve the goal of growing oocytes invitro.

Leptin treatment of in vitro cultured embryos increases outgrowth rate of inner cell mass during embryonic stem cell derivation

2019 ◽  
Vol 55 (7) ◽  
pp. 473-481 ◽  
Author(s):  
Ali Cihan Taskin ◽  
Ahmet Kocabay ◽  
Ayyub Ebrahimi ◽  
Sercin Karahuseyinoglu ◽  
Gizem Nur Sahin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Stem Cell Derivation

GENERATION OF RABBIT PLURIPOTENT STEM CELL LINES

2012 ◽  
Vol 24 (1) ◽  
pp. 286
Author(s):  
A. Dinnyes ◽  
M. K. Pirity ◽  
E. Gocza ◽  
P. Osteil ◽  
N. Daniel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Pluripotent Cells ◽  
Domestic Species ◽  
Isolation And Characterization ◽  
Induced Pluripotent ◽  
Pluripotency Genes

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate to all the somatic tissues. They can be genetically manipulated in vitro by knocking in and out genes, therefore they serve as an excellent tool for gene-function studies and for the generation of models for human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, several attempts have been made to generate pluripotent stem cells from other species as it would help us to understand the differences and similarities of signaling pathways involved in pluripotency and differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved among different species. This review gives an overlook of embryonic and induced pluripotent stem cell (iPSCs) research in the rabbit which is one of the most relevant non-rodent species for animal models. To date, several lines of putative ESCs and iPSCs have been described in the rabbit. All expressed stem cell-associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit full pluripotency in vivo. Moreover, similarly to several domestic species, markers used to characterize the putative ESCs are not fully adequate because studies in domestic species have revealed that they are not specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing rabbit embryo. The status of isolation and characterization of the putative pluripotency genes in rabbit will be discussed. Using rabbit specific pluripotency genes we might be able to reprogram somatic cells and generate induced pluripotent stem cells more efficiently thus overcome some of the challenges towards harnessing the potential of this technology. This study was financed by EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; RabPstem, PERG07-GA-2010-268422; PluriSys, HEALTH-2007-B-223485; AniStem, PIAP-GA-2011-286264), NKTH-OTKA-EU-7KP HUMAN-MB08-C-80-205; Plurabbit, OMFB-00130-00131/2010 ANR-NKTH/09-GENM-010-01.

Ex-Vivo Generation and Expansion of Both Lymphoid and Myeloid Lineages from Human Cord Blood (CB) HSC Using a Serum-Free Human Mesenchymal Stem Cell Based Culture System.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2888-2888
Author(s):  
Ana Frias ◽  
Christopher D. Porada ◽  
Kirsten B. Crapnell ◽  
Joaquim M.S. Cabral ◽  
Esmail D. Zanjani ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Cell Number ◽  
Human Cord Blood ◽  
Serum Free ◽  
Gm Csf ◽  
Cd34 Cells

Abstract The in vitro culture of a hematopoietic stem cell (HSC) graft with either media containing animal-derived components or a feeder layer with ill-defined pathogenic potential such as xenogeneic cell lines or cells modified by viral transformation poses risks that concern scientists and regulatory agencies. In the present studies, we avoided these risks by evaluating the ability of a human stromal-based serum free culture system (hu-ST) to support the ex-vivo expansion/maintenance of human CB HSC. CB CD34+ enriched cells were cultured in serum free medium in the presence of hu-ST with SCF, bFGF, LIF and Flt-3, and the cultures were analyzed for expansion, phenotype and clonogenic ability. We have previously reported the ability of this culture system to allow the successful expansion/maintenance of HSC along the myeloid pathway. In the present study, we investigated whether we could further develop this culture system to simultaneously expand myelopoiesis and lymphopoiesis in vitro. To this end, cord blood CD34+ cells were cultured for a total of 28 days and analyzed every 3 days for expansion and phenotype. There was a progressive increase in CD34 cell number with time in culture. The differentiative profile was primarily shifted towards the myeloid lineage with the presence of CD33, CD15, and CD14. However, a significant number of CD7+ cells were also generated. At week 2 of culture, we observed that 30% of the cells in the culture were CD7 positive. These CD7+CD2-CD3-CD5-CD56-CD16-CD34- cells were then sorted and either plated on top of new irradiated hu-ST layers in the presence of SCF, FLT-3, IL-7, IL-2, and IL-15, or cultured with IL-4, GM-CSF, and FLT-3 in the absence of stroma. Both of these cultures were maintained for an additional 2 weeks. In both sets of cultures, further expansion in the total cell number occurred with the time in culture, and by the end of the week 2, we observed that 25.3±4.18% of the cells had become CD56+ CD3-, a phenotype consistent with that of NK cells. Furthermore, cytotoxicity assays were performed and showed cytotoxic activity that increased in an E:T ratio-dependent fashion. 38.6% of the CD7+ cells grown in the presence of IL-4, GM-CSF, and FLT-3 became CD123+CD11c-, a phenotype characteristic of nonactivated dendritic cells, while 7.3–12.1% adopted an activitated dendritic cell phenotype CD83+CD1a+. In summary, we developed an in vitro culture system that reproducibly allows the effective ex vivo expansion of human cord blood HSCs while maintaining the capability of generating both myeloid and lymphoid hematopoiesis in vitro.

Morphologic, Immunophenotypic and in Vitro Growth Characteristics of Blood and Bone Marrow Associated with Stem Cell Mobilisation in Patients with Lymphoma

2000 ◽  
Vol 38 (3-4) ◽  
pp. 351-361 ◽  
Author(s):  
Michele Ghielmini ◽  
Giovanna Marangoni ◽  
Sandro Pampallona ◽  
Patricia Tamasy ◽  
Franco Cavalli
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Growth Characteristics ◽  
In Vitro Growth ◽  
Stem Cell Mobilisation

scholarly journals Interval in the replacement of in vitro culture medium affects the integrity and development of equine preantral follicles

2018 ◽  
Vol 38 (12) ◽  
pp. 2284-2288
Author(s):  
Camila Bizarro-Silva ◽  
Suellen M. González ◽  
Isabela Búfalo ◽  
Andressa G. Lindquist ◽  
Fabiana D. Sarapião ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture System ◽  
Culture Medium ◽  
Follicular Development ◽  
Significance Level ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Medium Exchange ◽  
Significant Difference

ABSTRACT: The efficiency of a culture system is related to the elaboration and replacement of a medium with conditions suitable for follicular development. Recent investigations suggested that in vitro culture medium should be replaced after specific time periods in various species. However, the suitable interval for the exchange of in vitro culture medium has not yet been established in equine species. The objective of this investigation was to evaluate the effect of medium exchange intervals of 24 hours (T24) or 48 hours (T48) for in vitro culture of preantral follicles at 2 or 6 days. At the end of the culture period, the fragments were processed using classical histology. Equine preantral follicles were classified according to morphological integrity and developmental stage. Data analysis was performed using Fisher’s test with a significance level of p<0.05. Out of a total of 399 follicles evaluated, 174 (43.6%) were primordial follicles, 225 (56.4%) were in development, and 63.76% were morphologically intact. In the in vitro culture performed over two days, there was no significant difference in relation to follicular integrity after medium replacement (p>0.05). Compared to the medium replacement at six days of culture, there was a statistically significant difference for T24 (68.9%, p<0.05). Therefore, we suggest changing the medium for equine species at 48 hours after the start of culture followed by subsequent daily replacements.

A Unique Pre-Clinical Model Providing Access to a Drug Resistant Population within the Multiple Myeloma Stem Cell Niche in a Novel 3-D Culture System for Bone Marrow.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 547-547
Author(s):  
Julia Kirshner ◽  
Kyle J. Thulien ◽  
Lorri D. Martin ◽  
Carina Debes Marun ◽  
Tony Reiman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Stem Cell ◽  
Culture System ◽  
Hematopoietic Cells ◽  
Drug Resistant ◽  
Culture Model

Abstract Bone marrow (BM), a site of hematopoiesis, is a multicellular tissue with a complex architecture. Multiple myeloma (MM) is an incurable plasma cell malignancy where even patients in remission succumb to an inevitable relapse. While considerable progress has been made towards understanding and treating MM, to date, there is no culture system which can recapitulate the complex interactions within the BM microenvironment. Current failure to grow the MM clone within the context of human microenvironment hampers progress into the understanding of the biology of MM and design of biologically relevant therapies. Here we present an in vitro three-dimensional (3-D) tissue culture model which recapitulates the human BM microenvironment allowing for the growth and expansion of the MM clone. Cells from the BM aspirates are grown in a fibronectin, laminin and collagen rich ECM designed to reconstruct in vitro endosteum and central marrow, mimicking the in vivo microenvironment of the BM. Proliferation and redistribution of cells within reconstructed ECM results in stratification of the culture, mimicking the in vivo condition where cells occupy individual niches. Cellular composition of the culture is maintained in accordance with the proliferation properties of the BM where osteoblasts, osteoclasts, adipocytes and stromal cells differentiate along with the full complement of the hematopoietic cells. BM cultures from normal donors are well-organized with osteoclasts and hematopoietic cells occupying distinct positions in the ECM. In contrast, reconstructed BM from MM patients is disorganized in 3-D where osteoclasts intermingle with the hematopoietic compartment. The MM malignant clone is expanded in 3-D cultures as measured by real-time quantitative PCR (rqPCR) for genomic clonotypic VDJ sequences. Malignant B and plasma cells proliferate in these cultures and FISH analysis reveals that their progeny harbor chromosomal abnormalities identical to those that mark the malignant clone prior to culture. Preclinical testing of emerging therapeutics targeted for multiple myeloma is hindered by the failure of the current models to sustain growth of the myeloma clone. In the 3-D culture, myeloma clone expands within its native environment providing an ideal preclinical model where conventional (Melphalan) and novel (Velcade) therapeutics efficiently and selectively kill their target cells. In the 3-D BM culture model, non-proliferating, label retaining cells (LRC) concentrate at a putative endosteum-marrow junction, where hematopoietic stem cells have been shown to localize in vivo, suggesting that the drug-resistant myeloma stem cells localize to the endosteal niche. In a colony-forming assay, drug-resistant LRC purified from the 3-D cultures form clonal colonies composed of malignant cells with patient specific clonotypic VDJ sequences. Recapitulation of the BM architecture in vitro is a first step towards the identification and therapeutic targeting of the elusive myeloma stem cell.

CD34+ Subclasses of Blood Stem Cell Grafts Collected After Plerixafor Injection in Non-Hodgkin Lymphoma (NHL) Patients Mobilizing Poorly with Chemotherapy Plus G-CSF

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2990-2990
Author(s):  
Esa Jantunen ◽  
Ville Varmavuo ◽  
Piia Valonen ◽  
Taru Kuittinen ◽  
Tapio Nousiainen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Stem Cell Mobilization ◽  
High Dose ◽  
In Vitro Growth ◽  
Control Groups ◽  
Cd34 Cells ◽  
Cell Mobilization

Abstract Abstract 2990 Background: Mobilization of blood stem cells is difficult in a subgroup of patients with standard methods. Plerixafor, a CXCR4 antagonist, has been used for stem cell mobilization in combination with G-CSF for some years. Mobilization method used may affect not only efficacy of stem cell mobilization and collection but also graft content which on the other hand may have effect on post-transplant outcomes. No data is available on CD34+ subclasses in grafts collected after plerixafor administration in patients who mobilize poorly with chemotherapy plus G-CSF. Patients and Methods: Altogether blood stem cell grafts collected from 26 NHL patients were studies. Thirteen patients (8 M, 5 F, median age 51 yrs) were mobilized with a combination of chemotherapy and G-CSF ad received plerixafor due to poor mobilization followed by stem cell apheressis. Thirteen patients (10 M, 3 F, median age 56 yrs) were mobilized with chemotherapy plus G-CSF without plerixafor and served as controls. Samples from the first collection after plerixafor and from the first apheresis of control patients were studied by flow cytometry using the following antibodies: CD34, CD38, CD 117, CD133, CD19 and CD45. Viability of CD34+ cells after freezing was assessed with 7-aminoactinomycin D staining. Also in vitro growth of granulocyte/macrophage progenitors (GM-CFU) were assessed from all grafts. Patients were followed after high-dose chemotherapy in regard to hematopoietic reconstitution. Results: The number of viable cells in the grafts was comparable between the plerixafor and the control groups (Table 1). The number of the most primitive stem cells (CD34+CD38−CD133+) was higher in plerixafor mobilized grafts (Table 1). Most of the CD34+ cells were myeloid progenitors, as defined by their CD117 antigen co-expression. No differences in GM-CFU were observed between the groups. All except one patient had received high-dose therapy. The median number of CD34+ cells collected from the patients was comparable (3.1 vs. 3.3 × 106/kg). The median time to reach neutrophils > 10 × 109/L was 10 days from the stem cell infusion in both groups and time to unsupported platelets was also comparable (16 d vs. 13 d). Platelet counts at 1 month, 3 months and 6 months were comparable between the groups. Absolute lymphocyte counts were higher in plerixafor group but the differences were not statistically significant. One early toxic death occurred in the plerixafor mobilized group and one death due to disease recurrence in both groups with a median follow-up of 301 and 348 days from stem cell infusion in prelixafor and control groups, respectively. Conclusions: Plerixafor added to chemomobilization in NHL patients resulted in higher number of the most primitive CD34+ cells in the graft with comparable in vitro growth and engraftment potential after BEAM chemotherapy when compared to patients mobilized without plerixafor. Longer follow-up of higher patient numbers are needed to evaluate whether differences in graft content have an effect on patient outcomes. Disclosures: Jantunen: Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Effect of stem cell factor on colony growth from acquired and constitutional (Fanconi) aplastic anemia

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 382-387 ◽  
Author(s):  
GP Bagnara ◽  
P Strippoli ◽  
L Bonsi ◽  
MF Brizzi ◽  
GC Avanzi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Aplastic Anemia ◽  
Stem Cell Factor ◽  
In Vitro Growth ◽  
Hematopoietic Progenitors ◽  
Gm Csf ◽  
Normal Controls ◽  
Marrow Cells

Abstract The aim of this study was to evaluate the effect of stem cell factor (SCF) on the in vitro growth of bone marrow hematopoietic progenitors from patients with acquired severe aplastic anemia (AA) or Fanconi's anemia (FA). For this purpose, we studied 11 patients with acquired AA (5 at diagnosis, 6 after ALG treatment), 12 patients with FA, and nine normal controls. Bone marrow cells were plated in vitro for colony- forming unit granulocyte-macrophage (CFU-GM) (in the presence of granulocyte-macrophage colony-stimulating factor [GM-CSF]), and for burst-forming unit-erythroid (BFU-E) and CFU-granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) colonies (in the presence of erythropoietin and interleukin-3 [IL-3]), with or without 20 ng/mL of SCF. In normal controls, SCF enhanced the growth of CFU-GM colonies from 103 to 263 (median), of BFU-E from 168 to 352, and of GEMM colonies from 6 to 38/10(5) cells plated. In patients with acquired AA, SCF induced a significant enhancement of BFU-E growth (8 to 29; P = .01) and allowed the formation of GEMM colonies that were not scored in baseline culture conditions (0 to 8; P = .01). CFU-GM growth was enhanced (4 to 20), but not significantly (P = .3). This was true both for patients at diagnosis and after antilymphocyte globulin treatment. By contrast, 10 of 12 FA patients grew no CFU-GM, BFU-E, or CFU-GEMM colonies, with or without SCF. In two FA patients (one transfusion- dependent and one transfusion-independent), an enhancement of CFU-GM and/or BFU-E was observed. The lack of response of hematopoietic progenitor cells from FA patients to GM-CSF+SCF or IL-3+SCF was not dependent on a defective expression of cytokine receptor messenger RNAs. Northern blot analysis showed in marrow cells from acquired AA and FA patients the presence of normal transcripts for alpha- and beta- chains of GM-CSF/IL-3 receptor and for c-kit protein. In conclusion, SCF promotes the in vitro growth of hematopoietic progenitors in patients with acquired AA, but not in patients with FA, pointing out the intrinsic nature of the defect in the latter disorder.

scholarly journals Effects of stem cell factor (kit-ligand) and interleukin-3 on the growth and serine proteinase expression of rat bone-marrow-derived or serosal mast cells

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 72-83 ◽  
Author(s):  
DM Haig ◽  
JF Huntley ◽  
A MacKellar ◽  
GF Newlands ◽  
L Inglis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mast Cells ◽  
Stem Cell Factor ◽  
Synergistic Effects ◽  
Serine Proteinase ◽  
In Vitro Growth ◽  
Interleukin 3 ◽  
Kit Ligand

Abstract The effects of rat stem-cell factor (SCF) and interleukin-3 (IL-3), alone or in combination, on the in vitro growth and serine proteinase expression of rat serosal/connective-tissue mast cells (CTMC) or bone marrow-derived mast cells (BMMC) were examined. Rat SCF stimulated the growth of both CTMC and BMMC. IL-3 stimulated BMMC growth to a lesser extent than did SCF, whereas CTMC numbers did not increase in IL-3. However, SCF and IL-3 had synergistic effects on the growth of both BMMC and CTMC. SCF favoured the maintenance of rat mast cell proteinase- I (RMCP-I) in CTMC, but did not induce detectable production of RMCP-I in BMMC. In contrast, when IL-3 or lymph node-conditioned medium (LNCM) was added to SCF, a subpopulation of CTMC expressed and stored the soluble proteinase RMCP-II. In BMMC, the RMCP-II content of cells maintained in SCF was significantly less than that of cells maintained in IL-3 or LNCM. RMCP-II also appeared in the supernatants of BMMC, especially when BMMC numbers were increasing rapidly in SCF with or without IL-3 or LNCM. Thus, SCF and IL-3 can regulate the growth of rat BMMC and CTMC, as well as influence their production and release of proteinases.

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