scholarly journals Co-growth of Stem Cells With Target Tissue Culture as an Easy and Effective Method of Directed Differentiation

2021 ◽  
Vol 9 ◽  
Author(s):  
Marina Valentinovna Kovina ◽  
Tatyana Gennadievna Dyuzheva ◽  
Mikhail Evgenievich Krasheninnikov ◽  
Sergey Alexandrovich Yakovenko ◽  
Yury Mikhailovich Khodarovich
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem ◽  
Cell Contact ◽  
Target Tissue ◽  
Hepatic Differentiation ◽  
Endothelial Surface ◽  
Adult Mesenchymal Stem Cells

The long-term co-culture of mouse embryonic stem cells (mESC) with rat endothelial cells (EC) was tested for contact differentiation into the endothelial lineage. Serial passaging of rat ECs mixed with mESC in ratio 10:1 resulted in the emergence of a homogeneous cell population expressing mouse endothelial surface markers CD102, CD29, CD31. Rat endothelial surface marker RECA-1 completely disappeared from the co-cultured population after 2 months of weekly passaging. Co-incubation of mESC with rat ECs without cell-to-cell contact did not result in the conversion of mESC into ECs. After co-cultivation of adult mesenchymal stem cells from human endometrium (eMSC) with pre-hepatocyte-like cells of human hepatocarcinoma Huh7 the resulting co-culture expressed mature liver markers (oval cell antigen and cytokeratin 7), none of which were expressed by any of co-cultivated cultures, thus proving that even an immature (proliferating) pre-hepatocyte-like line can induce hepatic differentiation of stem cells. In conclusion, we have developed conditions where long-term co-proliferation of embryonic or adult SC with fully or partially differentiated cells results in stem cell progeny expressing markers of target tissue. In the case of endothelial differentiation, the template population quickly disappeared from the resulted culture and the pure endothelial population of stem cell progeny emerged. This approach demonstrates the expected fate of stem cells during various in vivo SC-therapies and also might be used as an effective in vitro differentiation method to develop the pure endothelium and, potentially, other tissue types of desirable genetic background.

Effect of NANOG overexpression on porcine embryonic development and pluripotent embryonic stem cell formation in vitro

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Gerelchimeg Bou ◽  
Shimeng Guo ◽  
Jia Guo ◽  
Zhuang Chai ◽  
Jianchao Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Embryonic Stem Cell ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Cell Clones ◽  
Morula Stage ◽  
Pluripotent Embryonic Stem Cell

Summary The efficiency of establishing pig pluripotent embryonic stem cell clones from blastocysts is still low. The transcription factor Nanog plays an important role in maintaining the pluripotency of mouse and human embryonic stem cells. Adequate activation of Nanog has been reported to increase the efficiency of establishing mouse embryonic stem cells from 3.5 day embryos. In mouse, Nanog starts to be strongly expressed as early as the morula stage, whereas in porcine NANOG starts to be strongly expressed by the late blastocyst stage. Therefore, here we investigated both the effect of expressing NANOG on porcine embryos early from the morula stage and the efficiency of porcine pluripotent embryonic stem cell clone formation. Compared with intact porcine embryos, NANOG overexpression induced a lower blastocyst rate, and did not show any advantages for embryo development and pluripotent embryonic stem cell line formation. These results indicated that, although NANOG is important pluripotent factor, NANOG overexpression is unnecessary for the initial formation of porcine pluripotent embryonic stem cell clones in vitro.

Accept or Reject: The Role of Immune Tolerance in the Development of Stem Cell Therapies and Possible Future Approaches

2020 ◽  
pp. 019262332091824
Author(s):  
Richard Haworth ◽  
Michaela Sharpe
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem ◽  
Immune Recognition ◽  
Cell Of Origin ◽  
In Vivo Models ◽  
Cell Product ◽  
A Cell

In 2011, Goldring and colleagues published a review article describing the potential safety issues of novel stem cell-derived treatments. Immunogenicity and immunotoxicity of the administered cell product were considered risks in the light of clinical experience of transplantation. The relative immunogenicity of mesenchymal stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) was being addressed through in vitro and in vivo models. But the question arose as to whether the implanted cells needed to be identical to the recipient in every respect, including epigenetically, to evade immune recognition? If so, this set a high bar which may preclude use of many cells derived from iPSCs which have vestiges of a fetal phenotype and epigenetic memory of their cell of origin. However, for autologous iPSCs, the immunogenicity reduces once the surface antigen expression profile becomes close to that of the parent somatic cells. Therefore, a cell product containing incompletely differentiated cells could be more immunogenic. The properties of the administered cells, the immune privilege of the administration site, and the host immune status influence graft success or failure. In addition, the various approaches available to characterize potential immunogenicity of a cell therapy will be discussed.

396 Oct-4 EXPRESSION IN CAT INNER CELL MASS-DERIVED CELLS CULTURED IN MEDIUM WITH DIFFERENT PROTEIN SOURCES

2010 ◽  
Vol 22 (1) ◽  
pp. 354
Author(s):  
T. S. Rascado ◽  
J. F. Lima-Neto ◽  
S. E. R. S. Lorena ◽  
B. W. Minto ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Domestic Cat ◽  
Santa Cruz

The domestic cat can be used as a biological model for humans because of similarities in some disease and genetically transmitted conditions. Embryonic stem cells might complete nuclear reprogramming more efficiently than somatic cells and, therefore, are potentially useful for increasing interspecific cloning success. The objective of this study was to establish an effective culture system for inner cell mass (ICM)-derived cells in the domestic cat, testing the ability of the ICM to attach to the culture dish and to form embryonic stem cell colonies in the presence of fetal calf serum (FCS) and Knockout serum (KS). Moreover, knowing that the transcription factor Oct-4 is important for the maintenance of pluripotency in human and murine embryonic stem cells, the expression of this factor was evaluated in in vitro-produced blastocyst and in the attached ICM. Domestic cat oocytes were matured, fertilized, and cultured in vitro until the blastocyst stage. The ICM was mechanically isolated (n = 60) using a scalpel blade and transferred to a monolayer of chemically inactivated cat fibroblasts with 10 μg mL-1 mitomicin C. The base culture media (BM) was DMEM/F12 supplemented with nonessential amino acids, glutamine, leukemia inhibitory factor, fibroblast growth factor-2, 2-mercaptoethanol, and antibiotics. Three groups were tested: G1 = BM with 20% FCS (20); G2 = BM with 20% KS (20); G3 = BM with 15% FSC and 5% KS (20). Culture was performed in a 5% CO2 in air incubator at 38.5°C. No statistical difference was observed among groups in relation to ICM attachment (chi-square, P > 0.05). Ninety percent of the ICM presented good adhesion after 3 days of culture and started to grow in all media tested. However, until now, no good colonies were formed. Fifteen blastocysts and 10 attached ICM were fixed in 3% paraformaldehyde and permeabilized in 0.2% triton X-100 in PBS. Subsequently, to block nonspecific binding of the primary antibody, the preadsorption for 2 h at room temperature with OCT4 blocking peptide (sc-8628P, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. Samples were incubated with Oct4 antibody (N-19 : sc 8628, Santa Cruz Biotechnology) and with the appropriate secondary antibody (A21431, Invitrogen) and examined by fluorescence microscopy. Oct4 protein was detected both in the ICM and trophoderm cells, and it was distributed in cytoplasm and nuclei. These embryos were also stained with Hoechst 33342. Although further standardization of the culture media is needed, it seems that the KS can be replaced by FCS in cat embryonic stem cell culture. Furthermore, the immunostain of the trophoderm with Oct-4 indicates a difference in the expression of this factor when compared with its expression on human and murine blastocysts. This could be related to in vitro production, or Oct 4 is not a good pluripotency marker for cat embryos and cat embryonic stem cell, consequently. This fact has been noted in goat, bovine, and porcine embryos. Acknowledgment is given to FAPESP.

scholarly journals “Opening” The Mesenchymal Stem Cell Tool Box

2009 ◽  
Vol 03 (03) ◽  
pp. 240-249 ◽  
Author(s):  
Fares Zeidán-Chuliá ◽  
Mami Noda
Keyword(s):  
Stem Cells ◽  
Adult Stem Cells ◽  
Current Status ◽  
Tooth Replacement ◽  
Cell Based Therapy ◽  
Adult Mesenchymal Stem Cells ◽  
Wide Potential ◽  
New Strategies

ABSTRACTAdult mesenchymal stem cells (MSCs) are adherent stromal cells able to self-renew and differentiate into a wide variety of cells and tissues. MSCs can be obtained from distinct tissue sources and have turned out to be successfully manipulated in vitro. As adult stem cells, MSCs are less tumorigenic than their embryonic correlatives and posses another unique characteristic which is their almost null immunogenicity. Moreover, these cells seem to be immunosuppressive in vitro. These facts together with others became MSCs a promising subject of study for future approaches in bioengineering and cell-based therapy. On the other hand, new strategies to achieve long-term integration as well as efficient differentiation of these cells at the area of the lesion are still challenging, and the signalling pathways ruling these processes are not completely well characterized. In this review, we are going summarize the general landscape and current status of the MSC tool as well as their wide potential in tissue engineering, from neuronal to tooth replacement. Highlights and pitfalls for further clinical applications will be discussed. (Eur J Dent 2009;3:240-249)

scholarly journals 5-Azacytidine-Induced Cardiomyocyte Differentiation of Very Small Embryonic-Like Stem Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
XiaoLin Sun ◽  
HongXiao Li ◽  
Ye Zhu ◽  
Pei Xu ◽  
QiSheng Zuo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Ethical Issues ◽  
Troponin T ◽  
Embryonic Stem ◽  
Stem Cell Population ◽  
Pacemaker Activity ◽  
Stem Cell Markers ◽  
Seed Cells

The use of stem cells in generating cell-based pacemaker therapies for bradyarrhythmia is currently being considered. Due to the propensity of stem cells to form tumors, as well as ethical issues surrounding their use, the seed cells used in cardiac biological pacemakers have limitations. Very small embryonic-like stem cells (VSELs) are a unique and rare adult stem cell population, which have the same structural, genetic, biochemical, and functional characteristics as embryonic stem cells without the ethical controversy. In this study, we investigated the ability of rat bone marrow- (BM-) derived VSELs to differentiate in vitro into cardiomyocytes by 5-Azacytidine (5-AzaC) treatment. The morphology of VSELs treated with 10 μM 5-AzaC increased in volume and gradually changed to cardiomyocyte-like morphology without massive cell death. Additionally, mRNA expression of the cardiomyocyte markers cardiac troponin-T (cTnT) and α-sarcomeric actin (α-actin) was significantly upregulated after 5-AzaC treatment. Conversely, stem cell markers such as Nanog, Oct-4, and Sox2 were continuously downregulated posttreatment. On day 14 post-5-AzaC treatment, the positive expression rates of cTnT and α-actin were 18.41±1.51% and 19.43±0.51%, respectively. Taken together, our results showed that rat BM-VSELs have the ability to differentiate into cardiomyocytes in vitro. These findings suggest that VSELs would be useful as seed cells in exploring the mechanism of biological pacemaker activity.

scholarly journals Long-term In vitro Expansion Alters the Biology of Adult Mesenchymal Stem Cells

2008 ◽  
Vol 68 (11) ◽  
pp. 4229-4238 ◽  
Author(s):  
Reza Izadpanah ◽  
Deepak Kaushal ◽  
Christopher Kriedt ◽  
Fern Tsien ◽  
Bindiya Patel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adult Mesenchymal Stem Cells ◽  
In Vitro Expansion

Long Term Maintenance of Myeloid Leukemia Stem Cell-Like Populations Cultured with Mesenchymal Stromal Cells (MSC)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3546-3546
Author(s):  
Sawa Ito ◽  
A. John Barrett ◽  
Andre Larochelle ◽  
Nancy F. Hensel ◽  
Keyvan Keyvanfar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Culture Conditions ◽  
Myelogenous Leukemia ◽  
Cell Cycle Analysis ◽  
Remission Induction ◽  
Leukemia Stem Cell

Abstract Abstract 3546 Because MSC support the growth and the differentiation of normal hematopoietic stem cells we hypothesized that MSC might also support leukemia cells, in particular leukemia stem cells (LSC) in vitro. We cultured blast cells from patients with acute myelogenous leukemia (AML) in liquid medium to study persistence of stem-cell-like and differentiated leukemia cell populations by flow cytometry, with and without MSC and additional growth factors. Cryopresrerved peripheral blood mononuclear cells (PBMC) were obtained from 6 AML patients (mean Age 47, range 23–74). Leukemia blasts were isolated by sorting live (propidium iodide (PI)-negative) CD34+ lineage (CD2+, CD3+, CD14+ and CD19+) -negative cells using a FACS ARIA II cell sorter (BD). Sorted blasts (2.5 ×105 cells) were co-cultured with an equal number of irradiated MSC derived from healthy donor bone marrow in RPMI medium supplemented with 10% human serum, with or without a human cytokine (CYTO) mixture (50 ng/ml interleukin 3, 150 ng/ml stem cell factor, and 150ng/ml Flt-3 ligand). MSC were replenished every two weeks. The phenotype of cultured cells was analyzed weekly using fluorescently-conjugated monoclonal antibodies against CD34, CD38, and CD45, plus the lineage panel and a dead cell exclusion dye Cell cycle analysis with Hoeschst 33342 and Pyronin Y was performed on cells co-stained with CD34, CD45 and PI. Primary leukemia samples were phenotypically heterogeneous with respect to proportions of cells (co-)staining for CD34 and CD38 as previously reported: three samples showed CD34+CD38- predominance (LSC-like leukemia), and three were CD34+CD38+ (common myeloid progenitor (CMP)-like leukemia). LSC-like leukemia maintained viable CD34+CD38- cells for at least 6 weeks when co-cultured with MSC alone, in contrast to cultures with cytokines or medium only which showed rapid decline in the LSC populations and no prolonged maintenance of viable cells (p=0.0005) (Figure, left panel). CMP-like leukemia maintained their CD34+CD38+ phenotype when co-cultured with MSC alone but persistence of this subset was not significantly different from the other culture conditions (p=0.5) and no culture remained viable after 4 weeks (Figure, right panel). Cell cycle analysis showed that co-culture with MSC maintained CD34+ blasts in G0 significantly more than other culture conditions (P<0.0001). We conclude that MSC support the maintenance of a leukemia stem cell phenotype in a long- term (6 week) in vitro culture system. The differential capacity of MSC to support LSC- like and CMP- like leukemia may be associated with the different frequency of leukemia initiating cells within each leukemic blast population. NSG mice xenotranplant model experiments are ongoing to confirm this hypothesis. Co-culture of LSC with MSC represents a simple approach to maintain LSC in vitro and could be utilized to screen the drug targeting LSCs. Further study of the effect of MSC on LSC would elucidate a potential mechanism whereby the marrow microenvironment serves as a reservoir of persisting leukemia after remission induction chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Hepatic differentiation of pluripotent stem cells

2009 ◽  
Vol 390 (10) ◽  
Author(s):  
Komal Loya ◽  
Reto Eggenschwiler ◽  
Kinarm Ko ◽  
Malte Sgodda ◽  
Francoise André ◽  
...  
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Pluripotent Stem Cells ◽  
Ethical Issues ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hepatic Differentiation ◽  
Alternative Source ◽  
Organ Specific

Abstract In regenerative medicine pluripotent stem cells are considered to be a valuable self-renewing source for therapeutic cell transplantations, given that a functional organ-specific phenotype can be acquired by in vitro differentiation protocols. Furthermore, derivatives of pluripotent stem cells that mimic fetal progenitor stages could serve as an important tool to analyze organ development with in vitro approaches. Because of ethical issues regarding the generation of human embryonic stem (ES) cells, other sources for pluripotent stem cells are intensively studied. Like in less developed vertebrates, pluripotent stem cells can be generated from the female germline even in mammals, via parthenogenetic activation of oocytes. Recently, testis-derived pluripotent stem cells were derived from the male germline. Therefore, we compared two different hepatic differentiation approaches and analyzed the generation of definitive endoderm progenitor cells and their further maturation into a hepatic phenotype using murine parthenogenetic ES cells, germline-derived pluripotent stem cells, and ES cells. Applying quantitative RT-PCR, both germline-derived pluripotent cell lines show similar differentiation capabilities as normal murine ES cells and can be considered an alternative source for pluripotent stem cells in regenerative medicine.

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