scholarly journals ФЕНОТИПОВІ ТА МОРФОЛОГІЧНІ ЗМІНИ КУЛЬТУРИ КЛІТИН КІСТКОВОГО МОЗКУ ЩУРІВ В ПРОЦЕСІ ЇХ КУЛЬТИВУВАННЯ

2016 ◽  
Vol 18 (2(66)) ◽  
pp. 126-132
Author(s):  
A.I. Mazurkiewicz ◽  
V.V. Kovpak ◽  
O.S. Kovpak
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Morphological Changes ◽  
Embryonic Stem ◽  
In Vitro Cultivation ◽  
Rat Bone ◽  
Marrow Cells

Bone marrow is the only adult tissue which normally consists of immature undifferentiated and low differentiated cells which called stem cells and they are similar in structure to embryonic stem cells. But literature data analysis doesn't give an unambiguous answer regarding phenotypic and morphological changes of bone marrow cells culture of rats during their in vitro cultivation which necessitated further research.Investigate phenotypic and morphological changes of bone marrow cells culture of rats during their in vitro cultivation from first to fourth passage.We were used in these research bone marrow cells of rats from the first to the fourth passages. Microscopic analysis and evaluation morphological changes of bone marrow cells culture of rats during cultivation were carried out using inverted microscope Axiovert 40. Control of changes phenotype was performed by detecting CD markers (CD10, CD38, CD34, CD45, CD48, CD54, CD56, CD66e, CD96, CD227, CD326, pan–keratin). The evaluation was performed by the semi– quantitative method (H–Score).The research of primary culture of rat bone marrow cells showed that it morphologically heterogeneous, noted the small number of cells polygonal shape, surrounded by the fibroblast cells. During the cultivation cell culture becomes more homogenous at the expense of fibroblast–like cells. As a result of occurred the transition process from heterogeneous culture in zero passage to the most homogeneous culture in 4 passage. Immunophenotyping population of cell culture derived from rat bone marrow, revealed a high level of expression of pan–keratin; moderate level – CD34, CD48, CD66e, CD95; low level – CD38, CD45, CD56, CD227, CD326; lack of expression – CD10, CD54. Change of the expression of surface markers varies in each passage CD48, CD66e, CD95 increased significantly; CD38, SD45, SD326, pan–keratin reduced significantly. The markers CD34, CD 56, CD 227 were expressed on the one level from the first to the fourth passage. The expression of the CD10, CD54 markers during the study period was not identified.

scholarly journals THE PERSISTENCE OF HEMOPOIETIC STEM CELLS IN VITRO

1973 ◽  
Vol 56 (2) ◽  
pp. 429-433 ◽  
Author(s):  
Russell Meints ◽  
Eugene Goldwasser
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Hemopoietic Stem Cells ◽  
Slow Increase ◽  
Marrow Cells

Cells capable of forming colonies in spleens of irradiated mice (CFU) are lost temporarily when bone marrow cells from rats or mice are maintained in culture. Rat marrow CFU go through a minimum at about 3 days after which there is a slow increase in the number of CFU in culture, reaching a maximum at 9 days. Mouse marrow CFU reach a minimum at 3 days and a maximum at 7 days. Some rat marrow CFU persist in culture for as long as 28 days.

scholarly journals Differentiation of Adipose-Derived Mesenchymal Stem Cells into Neuron-Like Cells induced by using β-mercaptoethanol

2020 ◽  
Vol 17 (1(Suppl.)) ◽  
pp. 0235
Author(s):  
Maeda Mohammad ◽  
Ahmed Majeed Al-Shammari ◽  
Rafal H Abdulla ◽  
Aesar Ahmed ◽  
Aseel Khalid
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Light Chain ◽  
Bone Marrow Cells ◽  
Neurofilament Light Chain ◽  
Neurofilament Light ◽  
Study Objective ◽  
Marrow Cells

Background: Adipose derived-mesenchymal stem cells have been used as an alternative to bone marrow cells in this study. Objective: We investigated the in vitro isolation, identification, and differentiation of stem cells into neuron cells, in order to produce neuron cells via cell culture, which would be useful in nerve injury treatment. Method: Mouse adipose mesenchymal stem cells were dissected from the abdominal subcutaneous region. Neural differentiation was induced using β-mercaptoethanol. This study included two different neural stage markers, i.e. nestin and neurofilament light-chain, to detect immature and mature neurons, respectively. Results: The immunocytochemistry results showed that the use of β-mercaptoethanol resulted in the successful production of neuron cells. This was attributable to the increase and significant overexpression of the nestin protein during the early exposure period, which resulted in the expression of the highest levels of nestin. In comparison, the expression level of neurofilament light-chain protein also increased with time but less than nestin. Non-treated mesenchymal stem cells, considered as control showed very low expression for both markers. Conclusion: The results of this study indicate that adipose mesenchymal cells represent a good, easily obtainable source of bone marrow cells used to developing the differentiation process.

scholarly journals Effect of Zinc Supplementation on Renal Anemia in 5/6-Nephrectomized Rats and a Comparison with Treatment with Recombinant Human Erythropoietin

2019 ◽  
Vol 20 (20) ◽  
pp. 4985 ◽  
Author(s):  
Hui-Lin Feng ◽  
Yen-Hua Chen ◽  
Sen-Shyong Jeng
Keyword(s):  
Bone Marrow ◽  
Recombinant Human Erythropoietin ◽  
Zinc Supplementation ◽  
Bone Marrow Cells ◽  
Human Erythropoietin ◽  
Post Surgery ◽  
Rat Bone ◽  
Marrow Cells

Anemia is a severe complication in patients with chronic kidney disease (CKD). Treatment with exogenous erythropoietin (EPO) can correct anemia in many with CKD. We produced 5/6-nephrectomized rats that became uremic and anemic at 25 days post surgery. Injection of the anemic 5/6-nephrectomized rats with 2.8 mg zinc/kg body weight raised their red blood cell (RBC) levels from approximately 85% of the control to 95% in one day and continued for 4 days. We compared the effect of ZnSO4 and recombinant human erythropoietin (rHuEPO) injections on relieving anemia in 5/6-nephrectomized rats. After three consecutive injections, both the ZnSO4 and rHuEPO groups had significantly higher RBC levels (98 ± 6% and 102 ± 6% of the control) than the saline group (90 ± 3% of the control). In vivo, zinc relieved anemia in 5/6-nephrectomized rats similar to rHuEPO. In vitro, we cultured rat bone marrow cells supplemented with ZnCl2, rHuEPO, or saline. In a 4-day suspension culture, we found that zinc induced erythropoiesis similar to rHuEPO. When rat bone marrow cells were supplement-cultured with zinc, we found that zinc stimulated the production of EPO in the culture medium and that the level of EPO produced was dependent on the concentration of zinc supplemented. The production of EPO via zinc supplementation was involved in the process of erythropoiesis.

scholarly journals In vitro proliferation of primitive hemopoietic stem cells supported by stromal cells: evidence for the presence of a mechanism(s) other than that involving c-kit receptor and its ligand.

1992 ◽  
Vol 176 (2) ◽  
pp. 351-361 ◽  
Author(s):  
H Kodama ◽  
M Nose ◽  
Y Yamaguchi ◽  
J Tsunoda ◽  
T Suda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Hemopoietic Stem Cells ◽  
In Vitro Proliferation ◽  
Kit Receptor ◽  
Colony Forming Units ◽  
Marrow Cells

The preadipose cell line, PA6, can support long-term hemopoiesis. Frequency of the hemopoietic stem cells capable of sustaining hemopoiesis in cocultures of bone marrow cells and PA6 cells for 6 wk was 1/5.3 x 10(4) bone marrow cells. In the group of dishes into which bone marrow cells had been inoculated at 2.5 x 10(4) cells/dish, 3 of 19 dishes (16%) contained stem cells capable of reconstituting erythropoiesis of WBB6F1-W/Wv mice, indicating that PA6 cells can support the proliferation of primitive hemopoietic stem cells. When the cocultures were treated with an antagonistic anti-c-kit monoclonal antibody, ACK2, only a small number of day 12 spleen colony-forming units survived; and hemopoiesis was severely reduced. However, when the cocultures were continued with antibody-free medium, hemopoiesis dramatically recovered. To examine the proliferative properties of the ACK2-resistant stem cells, we developed a colony assay system by modifying our coculture system. Sequential observations of the development of individual colonies and their disappearance demonstrated that the stem cells having higher proliferative capacity preferentially survive the ACK2 treatment. Furthermore, cells of subclones of the PA6 clone that were incapable of supporting long-term hemopoiesis expressed mRNA for the c-kit ligand. These results suggest that a mechanism(s) other than that involving c-kit receptor and its ligand plays an important role in the survival and proliferation of primitive hemopoietic stem cells.

Hepatocyte Growth Factor Induces Differentiation of Adult Rat Bone Marrow Cells into a Hepatocyte Lineage in Vitro

2000 ◽  
Vol 279 (2) ◽  
pp. 500-504 ◽  
Author(s):  
Seh-Hoon Oh ◽  
Masahiro Miyazaki ◽  
Hirosuke Kouchi ◽  
Yusuke Inoue ◽  
Masakiyo Sakaguchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Bone Marrow Cells ◽  
Adult Rat ◽  
Hepatocyte Growth ◽  
Rat Bone ◽  
Marrow Cells

Effect of Normal and Neutropenic Dog Sera on Nucleic Acids of Rat Bone Marrow Cells in vitro*

1986 ◽  
Vol 33 (1-10) ◽  
pp. 123-130 ◽  
Author(s):  
G. A. Montes ◽  
A. S. Islas ◽  
E. Ocharan ◽  
J. A. Correa ◽  
W. G. Rudolph
Keyword(s):  
Bone Marrow ◽  
Nucleic Acids ◽  
Bone Marrow Cells ◽  
Rat Bone ◽  
Marrow Cells

In Vitro Myelo-Lymphoid Colony Formation by Mouse Hematopoietic Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3861-3861
Author(s):  
Jun Ooehara ◽  
Hina Takano ◽  
Shin-ichiro Takayanagi ◽  
Hiromitsu Nakauchi ◽  
Hideo Ema
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Culture Conditions ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Lymphoid Progenitors ◽  
Marrow Cells

Abstract Hematopoietic stem cells (HSCs) clonally differentiate into all myeloid, B-lymphoid, and T-lymphoid lineages. Mouse HSCs are known to form in vitro colonies comprised of morphologically identifiable myeloid cells such as neutrophils, macrophages, erythroblasts, and megakaryocytes. Whether HSCs are able to differentiate along B-and T-lymphoid lineages in such colonies remains obscure. The co-culture systems with stromal cells such as S17, OP9, OP9/Delta cells have been shown to support B- and T-cell development. These systems have been used to identify subclasses of progenitors with lymphoid potentials. However, neither B cells nor T cells have been successfully generated from HSCs in vitro. This is most likely due to the lack of culture conditions which support HSCs to differentiate into a certain stage of lymphoid progenitors. In this study, we attempted to use serum-free single-cell culture to identify cytokines which fill the developmental gap between HSCs and lymphoid progenitors. Here we show that myelo-lymphoid colonies are formed by HSCs in the presence of thrombopoietin (TPO), interleukin (IL)-11, or IL-12 together with stem cell factor (SCF). CD34-negative/low, c-Kit-positive, Sca-1-positive, lineage marker-negative (CD34-KSL) bone marrow cells were individually cultured with a combination of cytokines for 7 days. All cells in each colony were transplanted into each from a group of lethally irradiated mice, along with compromised bone marrow cells. The recipient mice were periodically analyzed after transplantation to detect transient myeloid and lymphoid reconstitution. All myeloid, B-, and T-lymphoid progenitor activities were detected in single colonies formed in the presence of SCF+TPO, SCF+IL-11, SCF+IL-12. Only myeloid progenitor activity was predominantly detected in single colonies formed in the presence of SCF+IL-3, consistent with previous observations in blast colony assays. All these combinations of cytokines support self-renewal in HSCs to varying degrees. We conclude that TPO, IL-11, and IL-12 directly act on HSCs and support them to differentiate into progenitors with lymphoid differentiation potential. Early differentiation pathways in HSCs are likely to be used in common by myeloid and lymphoid lineages and be supported in common by multiple cytokines.

Functional Hematopoietic Cells Derived From Mouse Embryonic Stem Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2304-2304
Author(s):  
Cheng Li ◽  
Daniel R. George ◽  
Nichole M. Havey ◽  
Jeffery M. Klco ◽  
Timothy J. Ley
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Embryonic Stem ◽  
Post Injection ◽  
Spleen Cells ◽  
Short Term ◽  
Marrow Cells

Abstract Abstract 2304 Despite two decades of effort, deriving long-term repopulating hematopoietic stem/progenitor cells (HSPCs) from embryonic stem cells (ESCs) has proven to be extremely difficult. Both embryoid body (EB)-based and stroma-based methods have been extensively explored. However, robust production of HSPCs from C57BL/6J-derived mouse ESCs (mESCs) has not yet been reported. Furthermore, in vivo engraftment of mES-derived HSCs (from any strain) has been achieved only with forced expression of HoxB4 or related oncogenes, which creates significant limitations for most studies. Here, we describe a stroma-based co-culture method to differentiate HSCs and progenitor populations from C57BL/6J-derived mESCs. After simple co-culture on OP9 stroma cells for one week, C57BL/6J-derived mESC lines differentiate into cells that mark as HSCs, CMPs, GMPs, and MEPs (by immunophenotyping); these cells are capable of giving rise to erythrocytes, monocytes, and mast cells (by morphology and immunophenotyping) after another week of culture in methylcellulose with hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). Similar in vitro hematopoietic differentiation has been achieved in several different C57BL/6J-derived mESCs (B6/Blu, B6-GFP, LK1, and B6 albino), B6/SVJ129 mESCs (R1), various SVJ129-derived mESCs (SWT16, EDJ22, and SCC10), and five independent C57BL/6J mouse embryonic fibroblast (MEF)-derived induced pluripotent stem cell (iPSC) lines. C57BL/6J ESCs derived from CAGGS-GFP transgenic mice (B6-GFP ESCs, which express high levels of GFP in all hematopoietic lineages) were used to determine whether we could obtain long-term engraftment of the OP9 differentiated ESCs. B6-GFP ESCs cultured for 7 days on OP9 cells were sorted by Kit+ surface staining. Sorted cells (1×105, 2×105, 4×105) were transferred into immunocompromised NSG mice via retro orbital injection (n=1 mouse per dose). Peripheral blood from the recipients injected with 2×105 and 4×105 cells showed 5% GFP positivity in the peripheral blood at weeks 12 and 16 post-transplant, while recipients injected with 1×105 cells had no detectable GFP+ cells in the periphery. Bone marrow cells and spleens were harvested at week 22. The recipient injected with 4×105 cells showed 5% GFP positivity in the bone marrow and 20% in the spleen. Engraftment was multi-lineage. Myeloid compartments (CD34+, CD11b+, Kit+, and Gr-1+) showed similar or less GFP positivity than overall bone marrow and spleen cells. Lymphoid (CD3+ and B220+) and erythroid (Ter119+) compartments also showed similar GFP positivity compared to overall bone marrow cells. However, lymphoid and erythroid compartments contained significantly higher GFP positivity (30–60%) than overall spleen cells. We have now modified the procedure to transfer 1×106 unfractionated B6-GFP ESCs grown for 7 days on OP9 stroma directly into NSG recipients. We have detected short-term engraftment 4 weeks post-injection in the peripheral blood of one recipient and multilineage splenic engraftment 8 weeks post-injection in two recipients, confirming that short-term repopulating cells are indeed generated by this method. Secondary transplants using the GFP+ bone marrow cells from the long-term engrafted mouse have been performed. This approach could be a valuable tool for studying the hematopoietic development of a variety of mESC lines, and potentially, iPSC lines as well. Disclosures: No relevant conflicts of interest to declare.

In Vitro Cultivation Technology of Rat Bone Marrow Mesenchymal Stem Cells

2019 ◽  
Vol 9 (1) ◽  
pp. 62-68
Author(s):  
Wei Li ◽  
Junjie Zeng ◽  
Ganghua Zhu ◽  
Yunpeng Dong ◽  
Dinghua Xie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
In Vitro Cultivation ◽  
Marrow Mesenchymal Stem Cells ◽  
Cultivation Technology ◽  
Rat Bone
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