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 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.

scholarly journals THE EFFECT OF HETEROLOGOUS ANTI-LYMPHOCYTE SERUM ON MOUSE HEMOPOIETIC STEM CELLS

1968 ◽  
Vol 127 (4) ◽  
pp. 731-748 ◽  
Author(s):  
Tom R. DeMeester ◽  
Norman D. Anderson ◽  
Charles F. Shaffer
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Function ◽  
Bone Marrow Cells ◽  
Hemopoietic Stem Cell ◽  
Hemopoietic Stem Cells ◽  
Marrow Cells

The present study has demonstrated that rabbit anti-mouse lymphocyte serum (RAMLS) has the capability of destroying bone marrow cells and suppressing hemopoietic stem cell function. The in vitro incubation of bone marrow suspensions with RAMLS caused extensive cell lysis with an apparent preferential destruction of lymphoid, erythroid, and blastoid elements. Using the spleen colony assay, the number of functional hemopoietic stem cells was found to be markedly reduced in bone marrow populations exposed to RAMLS in vitro. Further, this loss of stem cell function could be produced by exposing marrow suspensions to small concentrations of antiserum which did not produce detectable cytotoxic effects on the general marrow population. A similar effect of RAMLS upon hemopoietic stem cells was found in vivo. The intravenous injection of RAMLS into lethally irradiated mice immediately after the infusion of isogeneic marrow cells reduced the number of spleen colonies formed, indicating that the antiserum could exhibit a deleterious effect upon stem cells in the bloodstream of the intact animal. Normal animals treated with daily subcutaneous injections of RAMLS for 3 wk had a significantly reduced marrow content of functional hemopoietic stem cells, suggesting that RAMLS can affect stem cells located in situ in the bone marrow. The experiments indicate that RAMLS possesses potential marrow toxicity.

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 Demonstration of Thy-1 antigen on pluripotent hemopoietic stem cells in the rat.

1978 ◽  
Vol 148 (5) ◽  
pp. 1351-1366 ◽  
Author(s):  
I Goldschneider ◽  
L K Gordon ◽  
R J Morris
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Cell Activity ◽  
Hemopoietic Stem Cell ◽  
Hemopoietic Stem Cells ◽  
Stem Cell Activity ◽  
Series Of Experiments ◽  
Marrow Cells

Three approaches were used to demonstrate the presence of Thy-1 antigen on the surface of pluripotent hemopoietic stem cells in the rat. In the first, stem cells from fetal liver, neonatal spleen, and adult bone marrow were prevented from forming hemopoietic colonies in the spleens of irradiated recipients spleen (colony-forming unit assay) by incubation with antibodies to Thy-1 antigen. Highly specific rabbit heteroantiserum to purified rat brain Thy-1 antigen and mouse alloantisera to Thy-1.1-positive thymocytes were equally effective. This inhibition was neutralized by purified Thy-1 antigen. In a second series of experiments, Thy-1-positive and Thy-1-negative populations of nucleated bone marrow cells were separated by the FACS. All of the hemopoietic stem cell activity was recovered in the Thy-1-positive population. The stem cells were among the most strongly positive for Thy-1 antigen, being in the upper 25th percentile for relative fluorescence intensity. The relationships of Thy-1 antigen to the rat bone marrow lymphocyte antigen (BMLA) was shown in a third series of experiments. Rabbit anti-BMLA serum, which is raised against a null population of lymphocyte-like bone marrow cells, has been shown to have anti-stem cell activity. Here we demonstrate by double immunofluorescence, cocapping, and differential absorption studies that Thy-1 and BMLA are parts of the same molecule.

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.

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.

scholarly journals The Effect of Erythropoietin on Human Bone Marrow Cells In Vitro. I. Studies of Nine Cases of Bone Marrow Failure

Blood ◽  
1971 ◽  
Vol 37 (6) ◽  
pp. 624-633 ◽  
Author(s):  
HIDEAKI MIZOGUCHI ◽  
YASUSADA MIURA ◽  
FUMIMARO TAKAKU ◽  
SHIGERU SASSA ◽  
SHYOZO CHIBA ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Group A ◽  
In Vitro Response ◽  
Marrow Cells

Abstract The in vitro response to erythropoietin of bone marrow from nine patients with bone marrow failure were studied. Two types of patients were observed, those in which the marrow was responsive to erythropoietin and those which were nonresponsive. Ferrokinetic data corresponded well with the response to erythropoietin in vitro. In the nonresponsive group, a recovery of sensitivity to erythropoietin was observed when the patients improved clinically. The nature of the bone marrow failure was discussed in relation to erythropoietin and stem cells.

scholarly journals A limited role for p16Ink4a and p19Arf in the loss of hematopoietic stem cells during proliferative stress

Blood ◽  
2005 ◽  
Vol 106 (3) ◽  
pp. 827-832 ◽  
Author(s):  
Lilia Stepanova ◽  
Brian P. Sorrentino
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Dominant Role ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Serial Transplantation ◽  
Marrow Cells

Abstract It has long been known that prolonged culture or serial transplantation leads to the loss of hematopoietic stem cells (HSCs); however, the mechanisms for this loss are not well understood. We hypothesized that expression of p16Ink4a or p19Arf or both may play a role in the loss of HSCs during conditions of enhanced proliferation, either in vitro or in vivo. Arf was not expressed in freshly isolated HSCs from adult mice but was induced in phenotypically primitive cells after 10 to 12 days in culture. When cultured bone marrow cells from either Arf–/– or Ink4a-Arf–/– mice were compared to wild-type cells in a competitive repopulation assay, no significant differences in HSC activity were seen. We then evaluated the role of p19Arf and p16Ink4a in the loss of HSCs during serial transplantation. Bone marrow cells from Ink4a-Arf–/–, but not Arf–/–, mice had a modestly extended life span and, on average, supported reconstitution of one additional recipient compared to wild-type cells. Mice given transplants of Ink4a-Arf–/–cells eventually did die of hematopoietic failure in the next round of transplantation. We conclude that mechanisms independent of the Ink4a-Arf gene locus play a dominant role in HSC loss during conditions of proliferative stress.

scholarly journals Isolation of murine pluripotent hemopoietic stem cells.

1984 ◽  
Vol 159 (6) ◽  
pp. 1576-1590 ◽  
Author(s):  
J W Visser ◽  
J G Bauman ◽  
A H Mulder ◽  
J F Eliason ◽  
A M de Leeuw
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Indirect Evidence ◽  
Fluorescein Isothiocyanate ◽  
Cell Types ◽  
Mouse Bone Marrow ◽  
Hemopoietic Stem Cells ◽  
Morphological Criteria

A method described to purify pluripotent hemopoietic stem cells ( PHSC ) from adult mouse bone marrow. The method consists of three separation steps. First, bone marrow cells are centrifuged in a discontinuous metrizamide gradient and simultaneously labeled with wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC). Second, the low density cells are analyzed by a fluorescence-activated cell sorter (FACS) and the WGA-positive cells with medium forward and low perpendicular light scatter intensities are sorted. The WGA-FITC is removed from the cells by incubation with N-acetyl-D-glucosamine. Finally, the sorted cells are incubated with anti-H-2K-biotin and avidin-FITC and sorted a second time to enrich cells with high H-2K density. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 d and 6.6 colonies per 100 cells at 12 d after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony-forming unit/spleen) was 135 (range, 90--230; n = 15) and was similar to that for the cell type that provides radioprotection (180 +/- 70), indicating that these functional properties were copurified. Indirect evidence suggests that the spleen-seeding efficiency (f factor) of these cells is 0.10 and, therefore, the average purity of the sorted PHSC was 65% (range in 15 experiments, 35--110%). The sorted cells were all in the G1 or G0 phase of the cell cycle. They appeared to be undifferentiated blasts by morphological criteria. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. 32% of the sorted cells could be induced to form myeloid progeny in vitro. This procedure should be useful for direct studies on the regulation of hemopoietic cell differentiation.

High Level Expression of a Membrane-Anchored Inhibitor of HIV Infection in Murine Hematopoietic Stem and Progenitor Cells Does Not Pertubate Serial Multilineage Repopulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1758-1758
Author(s):  
Axel Schambach ◽  
Bernhard Schiedlmeier ◽  
Jens Bohne ◽  
Dorothee von Laer ◽  
Geoff Margison ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hiv 1 ◽  
Marrow Cells

Abstract T20 is a 36-amino-acid peptide that binds to HIV-1 gp41 and thereby acts as a fusion inhibitor, thus mediating potent and selective inhibition of HIV-1 entry in vitro and in vivo. An extended peptide expressed as an artificial, membrane-bound molecule (mbC46) efficiently inhibits HIV infection of primary human T-cells following retroviral vector mediated gene transfer (Egelhofer et al., J Virol, 2004). To develop an even more stringent approach to HIV gene therapy, we targeted hematopoietic stem cells. In 3 experimental groups of C57BL/6 mice (9 animals/group), we investigated the long-term toxicity of murine bone marrow cells transduced with M87o, a therapeutic vector designed to coexpress mbC46 and an HIV-derived RNA RRE-decoy to inhibit HIV replication. As controls we used the same vector containing an inactive C46 peptide and mock-transduced cells. Blood samples were collected monthly. Donor chimerism and transgene expression in multiple lineages were determined by FACS analysis and transgene integration was measured by real time PCR. Six months after transplantation, 4 mice per group were sacrificed and the remaining 5 mice per group were observed for another 6 months. In addition to the parameters mentioned above, we performed complete histopathology, blood counts and clinical biochemistry. Donor chimerism in all groups ranged from 82 – 94% (day 190 and day 349). In the M87o group, 60% of donor cells expressed mbC46. FACS data showed persisting transgene expression in T-cells (CD4, CD8, 65%), B-cells (B220, 46%), myeloid cells (CD11b, 68%), platelets (CD41, 19%), and RBC (60%) of the peripheral blood and bone marrow cells. Highly sustained gene marking (2–4 copies/genome) was noticed on day 190. To reveal latent malignant clones potentially originating from side effects of the genetic manipulation, 1x106 bone marrow cells from 4 primary recipients were transplanted into lethally irradiated secondary recipients (3 recipients/primary mouse) and these mice were observed for 8 months. All together, we could not observe any evidence for leukemogenic capacity. Analysis of peripheral blood and bone marrow showed a similar transgene expression pattern compared to the primary mice. To generate a complete chimerism of transgenic cells, we chose the human drug resistance gene methylguanine-methyltransferase (MGMT, P140K) to select for mbC46-transduced stem cells in vitro and in vivo. Different coexpression strategies were tested. Function of the MGMT protein was confirmed in a quantitative alkyltransferase assay and in a cytotoxicity assay using BCNU or temozolomide. In vitro selection of transduced 32D and PM1 cells with benzylguanine and BCNU showed >95% positive cells with evidence of polyclonal survival. Transduced PM1 cells underwent an HIV challenge assay. In vivo experiments in a murine bone marrow transplantation setting are ongoing to determine the potency and safety of combined retroviral expression of mbC46 and MGMT in relevant preclinical models. Successful conclusion of these studies will hopefully result in a phase I clinical trial testing the concept of generating an HIV-resistant autologous hematopoiesis.

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