scholarly journals Prolonged hematopoiesis in a primate bone marrow culture system: characteristics of stem cell production and the hematopoietic microenvironment

Blood ◽  
1979 ◽  
Vol 54 (4) ◽  
pp. 775-793
Author(s):  
MA Moore ◽  
AP Sheridan ◽  
TD Allen ◽  
TM Dexter
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Culture System ◽  
Bone Marrow Culture ◽  
Allogeneic Bone ◽  
Cellular Interactions ◽  
Suspension Cells ◽  
Cell Production ◽  
Hematopoietic Microenvironment

Maintenance of myelopoiesis and pluripotential stem cell production for prolonged periods in vitro hitherto has been limited to mouse bone marrow culture. In an effort to adapt the system for use in higher species, particularly in human and non-human primates, studies were undertaken using the prosimian species, Tupaia glis (tree shrew). In a number of experiments the duration of sustained normal hematopoiesis observed in cultures of this species, following a single inoculum of 5 X 10(6)--10(7) bone marrow cells, with or without addition of fresh allogeneic bone marrow exceeded 1 yr. Analysis of suspension cells obtained by weekly demidepopulation of such cultures revealed production of CFU-C, differentiating neutrophils, and basophils at high levels. Direct comparison with murine cultures indicated that in both species a complex series of cellular interactions takes place within an adherent environment of marrow-derived endothelial cells, macrophages, and fat-containing cells. Certain functional and ultrastructural features served to distinguish murine from Tupaia marrow cultures, and the prolonged duration of in vitro hematopoiesis in the latter species could be attributed to a regenerative capacity possessed by its adherent hematopoietic microenvironment. The availability of this primate marrow culture system should facilitate studies of hematopoiesis, viral leukemogenesis, and transplantation biology, which have more direct relevance to man than that provided by the existing murine system.

scholarly journals Prolonged hematopoiesis in a primate bone marrow culture system: characteristics of stem cell production and the hematopoietic microenvironment

Blood ◽  
1979 ◽  
Vol 54 (4) ◽  
pp. 775-793 ◽  
Author(s):  
MA Moore ◽  
AP Sheridan ◽  
TD Allen ◽  
TM Dexter
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Culture System ◽  
Bone Marrow Culture ◽  
Allogeneic Bone ◽  
Cellular Interactions ◽  
Suspension Cells ◽  
Cell Production ◽  
Hematopoietic Microenvironment

Abstract Maintenance of myelopoiesis and pluripotential stem cell production for prolonged periods in vitro hitherto has been limited to mouse bone marrow culture. In an effort to adapt the system for use in higher species, particularly in human and non-human primates, studies were undertaken using the prosimian species, Tupaia glis (tree shrew). In a number of experiments the duration of sustained normal hematopoiesis observed in cultures of this species, following a single inoculum of 5 X 10(6)--10(7) bone marrow cells, with or without addition of fresh allogeneic bone marrow exceeded 1 yr. Analysis of suspension cells obtained by weekly demidepopulation of such cultures revealed production of CFU-C, differentiating neutrophils, and basophils at high levels. Direct comparison with murine cultures indicated that in both species a complex series of cellular interactions takes place within an adherent environment of marrow-derived endothelial cells, macrophages, and fat-containing cells. Certain functional and ultrastructural features served to distinguish murine from Tupaia marrow cultures, and the prolonged duration of in vitro hematopoiesis in the latter species could be attributed to a regenerative capacity possessed by its adherent hematopoietic microenvironment. The availability of this primate marrow culture system should facilitate studies of hematopoiesis, viral leukemogenesis, and transplantation biology, which have more direct relevance to man than that provided by the existing murine system.

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.

scholarly journals Bone marrow-on-a-chip: Emulating the human bone marrow

2018 ◽  
Author(s):  
Stefan Sieber ◽  
Annika Winter ◽  
Johanna Wachsmuth ◽  
Rhiannon David ◽  
Maria Stecklum ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Culture System ◽  
3D Culture ◽  
In Vitro Cultivation ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem

AbstractMultipotent hematopoietic stem and progenitor cells HSPC reside in specialized stem cell niches within the bone marrow, that provide a suitable microenvironment for lifelong maintenance of the stem cells. Meaningful in vitro models recapitulating the in vivo stem cell niche biology can be employed for both basic research as well as for applied sciences and represent a powerful tool to reduce animal tests in preclinical studies. Recently we published the generation of an in vitro bone marrow niche model, capable of long-term cultivation of HSC based on an organ-on-a-chip platform. This study provides a detailed analysis of the 3D culture system including matrix environment analysis by SEM, transcriptome analysis and system intrinsic differentiation induction. Furthermore, the bone marrow on a chip model can serve to multiply and harvest HSPC, since repeated cell removal not compromised the functionality of the culture system. The prolongation of the culture time to 8 weeks demonstrate the capacity to apply the model in repeated drug testing experiments. The quality of the presented system is emphasized by the differentiation capacity of long-term cultivated HSPC in vitro and in vivo. Transplanted human HSPC migrated actively into the bone marrow of irradiated mice and contributed to the long-term reconstitution of the hematopoietic system after four and eight weeks of in vitro cultivation.The introduced system offers a multitude of possible applications to address a broad spectrum of questions regarding HSPC, the corresponding bone marrow niche biology, and pathological aberrations.

In Vitro T Cell Differentiation from Human Hematopoietic Stem Cells (HSC) and Lymphoid Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4159-4159
Author(s):  
Qian-Lin Hao ◽  
Yuhua Zhu ◽  
Lora Barsky ◽  
Ewa Zielinska ◽  
Mary A. Price ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Culture System ◽  
Cell Maturation ◽  
Cell Production ◽  
Cell Commitment ◽  
T Cell Maturation

Abstract The study of human thymopoiesis has been hampered by our inability to provide all the signals of the normal thymic microenvironment to human stem and progenitor cells in vitro. Although T cell production from CD34+ progenitors can be achieved in vitro, purified populations of the immature CD34+CD38- cells (which include HSC) do not generate T cells reliably with the existing assays. The most commonly cited system for human T cell assay, Fetal thymic organ culture (FTOC) from immune deficient mice, is logistically very difficult and low seeding efficiency precludes its use for clonal assays. Recently, a murine bone marrow stromal line engineered to constitutively express Delta-1 ligand (OP9-DL1 stroma), has been shown to support production of mature murine T cells from HSC [Schmitt, 2002] and ES cells [Schmitt, 2004]. Here we report the use of modifications to the OP9-DL1 system that allow assay of human T lymphopoiesis from HSC and progenitors. CD34+lin-CD38- cells (HSC) from cord blood (CB) and bone marrow (BM)cultured on OP9 control stroma (that does not express DL) produced CD19+ B cells and CD56+ NK cells and maintained CD34+ cells for several weeks. However, T cell commitment was not evident in these control cultures. In contrast, culture of HSC on OP9-DL1 stroma (Dr. Zuniga-Pflucker) resulted in robust growth of T cell precursors (as shown by expression of CD7 and cytoplasmic (cy) CD3 in over 50% of cells) and mRNA for pre-Tα. NK cells were also increased on OP9-DL1 relative to control OP9 stroma but B cell production was lost. Although T cell commitment to the CD34+CD7+ and CD34-cyCD3+CD7+ cells stages was accomplished on OP9-DL1 stroma, markers of further T cell maturation such as surface CD3, CD4 and CD8, were absent on the cells produced. Multiple combinations of growth factors (IL-7, IL-2, IL-15, IL-3, ckit ligand (KL) and thrombopoietin (Tpo)) to the OP9-DL1 stromal cultures failed to achieve further T cell maturation, although the combination of KL, Tpo, IL-7 produced optimal cell growth. However, when conditioned medium collected from human thymic stromal cultures (thyCM) (medium collected from adherent cells derived from human postnatal thymus and filtered through 0.45u) was added to OP9-DL1 co-cultures, cyCD3+ cells were increased and differentiation of CD3+CD4+ cells was achieved. Thymic CD34+lin-CD7+ cells produced large numbers of CD3+CD4+CD8+, CD4+CD8- and CD4-CD8+ cells in this system. We have now used the OP9-DL1/thyCM system to demonstrate the T cell potential of CB and BM CD34+CD38-lin-CD7- (HSC) and CB CD34+CD38-lin-CD7+ (CLP). In addition, efficient cloning of single CB HSC and primitive thymic progenitors has been achieved using this culture system. The data suggests that, although DL-1 is required and sufficient for early T lymphoid commitment, other factor(s) specific to human thymic stroma are required for production of more mature T cells; these factors can now be identified using the OP9-DL1 system. The availability of a simple monolayer culture system that allows T lymphoid commitment from HSC and progenitors and further differentiation to mature T cells will be of great value in the study of the lineage potential of progenitor populations and the regulation of human thymopoiesis.

scholarly journals Cell Source-Dependent In Vitro Chondrogenic Differentiation Potential of Mesenchymal Stem Cell Established from Bone Marrow and Synovial Fluid of Camelus dromedarius

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1918
Author(s):  
Young-Bum Son ◽  
Yeon Ik Jeong ◽  
Yeon Woo Jeong ◽  
Mohammad Shamim Hossein ◽  
Per Olof Olsson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Synovial Fluid ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Chondrocyte Differentiation ◽  
Differentiation Potential ◽  
Proliferation Capacity

Mesenchymal stem cells (MSCs) are promising multipotent cells with applications for cartilage tissue regeneration in stem cell-based therapies. In cartilage regeneration, both bone marrow (BM-MSCs) and synovial fluid (SF-MSCs) are valuable sources. However, the cellular characteristics and chondrocyte differentiation potential were not reported in either of the camel stem cells. The in vitro chondrocyte differentiation competence of MSCs, from (BM and SF) sources of the same Camelus dromedaries (camel) donor, was determined. Both MSCs were evaluated on pluripotent markers and proliferation capacity. After passage three, both MSCs showed fibroblast-like morphology. The proliferation capacity was significantly increased in SF-MSCs compared to BM-MSCs. Furthermore, SF-MSCs showed an enhanced expression of transcription factors than BM-MSCs. SF-MSCs exhibited lower differentiation potential toward adipocytes than BM-MSCs. However, the osteoblast differentiation potential was similar in MSCs from both sources. Chondrogenic pellets obtained from SF-MSCs revealed higher levels of chondrocyte-specific markers than those from BM-MSCs. Additionally, glycosaminoglycan (GAG) content was elevated in SF-MSCs related to BM-MSCs. This is, to our knowledge, the first study to establish BM-MSCs and SF-MSCs from the same donor and to demonstrate in vitro differentiation potential into chondrocytes in camels.

Atelocollagen Sponge as a Stem Cell Implantation Scaffold for the Treatment of Scarred Vocal Folds

2010 ◽  
Vol 119 (11) ◽  
pp. 805-810 ◽  
Author(s):  
Satoshi Ohno ◽  
Shigeru Hirano ◽  
Ichiro Tateya ◽  
Shin-Ichi Kanemaru ◽  
Hiroo Umeda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Fluorescent Protein ◽  
In Vitro Study ◽  
Vocal Folds ◽  
Green Fluorescent ◽  
Cell Implantation

Objectives: Treatment of vocal fold scarring remains a therapeutic challenge. Our group previously reported the efficacy of treating injured vocal folds by implantation of bone marrow—derived stromal cells containing mesenchymal stem cells. Appropriate scaffolding is necessary for the stem cell implant to achieve optimal results. Terudermis is an atelocollagen sponge derived from calf dermis. It has large pores that permit cellular entry and is degraded in vivo. These characteristics suggest that this material may be a good candidate for use as scaffolding for implantation of cells. The present in vitro study investigated the feasibility of using Terudermis as such a scaffold. Methods: Bone marrow—derived stromal cells were obtained from GFP (green fluorescent protein) mouse femurs. The cells were seeded into Terudermis and incubated for 5 days. Their survival, proliferation, and expression of extracellular matrix were examined. Results: Bone marrow—derived stromal cells adhered to Terudermis and underwent significant proliferation. Immunohistochemical examination demonstrated that adherent cells were positive for expression of vimentin, desmin, fibronectin, and fsp1 and negative for beta III tubulin. These findings indicate that these cells were mesodermal cells and attached to the atelocollagen fibers biologically. Conclusions: The data suggest that Terudermis may have potential as stem cell implantation scaffolding for the treatment of scarred vocal folds.

Evaluation of Congenital Neutropenic Disorders by in Vitro Bone Marrow Culture

PEDIATRICS ◽  
1977 ◽  
Vol 59 (5) ◽  
pp. 739-748
Author(s):  
Peter M. Falk ◽  
Kenneth Rich ◽  
Stephen Feig ◽  
E. Richard Stiehm ◽  
David W. Golde ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Severe Disease ◽  
Stem Cell Differentiation ◽  
Peripheral Blood Cell ◽  
Clinical Expression ◽  
Essentially Normal ◽  
Cell Counts

The congenital neutropenias are a heterogeneous group of diseases whose etiology and pathogenesis are largely unknown. We studied nine neutropenic patients from seven families. Evaluation included peripheral blood cell and differential cell counts, epinephrine and typhoid vaccine stimulation studies, Rebuck skin windows, and bone marrow aspirations for morphological assessment and for in vitro culture in liquid suspension and in agar plates. Parallel cultures were set up with and without colony-stimulating activity (CSA), and peripheral leukocytes were assayed for cellular production of CSA. Patients were initially classified on the basis of their clinical course: benign, mild, moderately severe, or severe disease. One patient in the moderately severe group had an immunoglobulin disorder. Morphologically normal mature granulocytes were seen in bone marrow aspirates of two patients, and maturational defects of varying degree were seen in the remaining seven. Colony formation in agar was markedly reduced below normal in three of seven, moderately reduced in two of seven, and greater than normal in two patients. Colonies in six of seven patients consisted exclusively of macrophages. Marrow from all but one of the nine patients demonstrated poor neutrophil development in suspension culture, and addition of CSA did not result in augmented granulocytic proliferation or maturation. A scheme of normal neutrophil maturation is proposed, and the nine patients were categorized according to this scheme. Four patterns of congenital neutropenia emerged: type 1 was the most benign form of disease with essentially normal clinical and in vitro parameters, and a defect considered to be due to a small committed stem cell pool, abnormal release, or excessive utilization peripherally; type 2 had mild disease with presumed defective committed stem cell differentiation along the granulocyte line; type 3 included benign to severe clinical expression with an apparent defect at the level of the committed granulocyte precursor more severe than in type 2; type 4 disease had varied clinical expression but evidence for a defect at the level of the pluripotent stem cell.

Sign in / Sign up

Export Citation Format

Share Document