Isolation of Nucleated Cells from Bone Marrow Aspirate v1

2021 ◽  
Author(s):  
Steven B. Wells ◽  
Peter A. Szabo ◽  
Nora Lam
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Bone Marrow Aspirate ◽  
Myeloid Cells ◽  
Cell Suspensions ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Dilution Factor ◽  
Density Centrifugation ◽  
Defined Media

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human bone marrow aspirate. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Isolation of Nucleated Cells from Whole Blood v1

2021 ◽  
Author(s):  
Steven B. Wells ◽  
Peter A. Szabo ◽  
Nora Lam
Keyword(s):  
Single Cell ◽  
Whole Blood ◽  
Myeloid Cells ◽  
Cell Suspensions ◽  
Dilution Factor ◽  
Human Whole Blood ◽  
Density Centrifugation ◽  
Defined Media

This protocol describes a method for the isolation of pan-lymphocytes and pan-myeloid cells from human whole blood. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of Single Cell Suspension from Human Lymph Node Tissue v1

2021 ◽  
Author(s):  
Steven B. Wells ◽  
Peter A. Szabo ◽  
Nora Lam ◽  
Maya M.L. Poon
Keyword(s):  
Lymph Node ◽  
Cell Suspension ◽  
Single Cell ◽  
Myeloid Cells ◽  
Cell Suspensions ◽  
Dilution Factor ◽  
Single Cell Suspension ◽  
Density Centrifugation ◽  
Defined Media ◽  
Human Lymph Node

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human lymph node tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of Single Cell Suspension from Human Spleen Tissue v1

2021 ◽  
Author(s):  
Steven B. Wells ◽  
Peter A. Szabo
Keyword(s):  
Cell Suspension ◽  
Single Cell ◽  
Myeloid Cells ◽  
Cell Suspensions ◽  
Dilution Factor ◽  
Single Cell Suspension ◽  
Human Spleen ◽  
Spleen Tissue ◽  
Density Centrifugation ◽  
Defined Media

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human spleen tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

scholarly journals Strategies to Identify Mesenchymal Stromal Cells in Minimally Manipulated Human Bone Marrow Aspirate Concentrate Lack Consensus

2021 ◽  
pp. 036354652199378
Author(s):  
Severin Ruoss ◽  
J. Todd Walker ◽  
Chanond A. Nasamran ◽  
Kathleen M. Fisch ◽  
Conner J. Paez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Bone Marrow Aspirate ◽  
Transcriptional Profile ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Background: There is a need to identify and quantify mesenchymal stromal cells (MSCs) in human bone marrow aspirate concentrate (BMAC) source tissues, but current methods to do so were established in cultured cell populations. Given that surface marker and gene expression change in cultured cells, it is doubtful that these strategies are valid to quantify MSCs in fresh BMAC. Purpose: To establish the presence, quantity, and heterogeneity of BMAC-derived MSCs in minimally manipulated BMAC using currently available strategies. Study Design: Descriptive laboratory study. Methods: Five published strategies to identify MSCs were compared for suitability and efficiency to quantify clinical-grade BMAC-MSCs and cultured MSCs at the single cell transcriptome level on BMAC samples being used clinically from 15 orthopaedic patients and on 1 cultured MSC sample. Strategies included (1) the guidelines by the International Society for Cellular Therapy (ISCT), (2) CD271 expression, (3) the Ghazanfari et al transcriptional profile, (4) the Jia et al transcriptional profile, and (5) the Silva et al transcriptional profile. Results: ISCT guidelines did not identify any MSCs in BMAC at the transcriptional level and only 1 in 9 million cells at the protein level. Of 12,850 BMAC cells, 9 expressed the CD271 gene. Only 116 of 396 Ghazanfari genes were detected in BMAC, whereas no cells expressed all of them. No cells expressed all Jia genes, but 25 cells expressed at least 13 of 22. No cells expressed all Silva genes, but 19 cells expressed at least 8 of 23. Most importantly, the liberalized strategies tended to identify different cells and most of them clustered with immune cells. Conclusion: Currently available methods need to be liberalized to identify any MSCs in fresh human BMAC and lack consensus at the single cell transcriptome and protein expression levels. These different cells should be isolated and challenged to establish phenotypic differences. Clinical Relevance: This study demonstrated that improved strategies to quantify MSC concentrations in BMAC for clinical applications are urgently needed. Until then, injected minimally manipulated MSC doses should be reported as rough estimates or as unknown.

Preparation of Single Cell Suspensions of the Intra-Epithelial Layer and Lamina Propria from Human Intestinal Tissue v1

2021 ◽  
Author(s):  
Steven B. Wells ◽  
Pranay Dogra ◽  
Josh Gray ◽  
Peter A. Szabo ◽  
Daniel Caron ◽  
...  
Keyword(s):  
Single Cell ◽  
Lamina Propria ◽  
Intestinal Tract ◽  
Distal Colon ◽  
Cell Suspensions ◽  
Epithelial Layer ◽  
Dilution Factor ◽  
Human Gut ◽  
Intestinal Tissue ◽  
Defined Media

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from the epithelial layer and the lamina propria of human gut sections of about one gram of tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples. This protocol can be used for any section of the intestinal tract from duodenum to distal colon.

scholarly journals Cytochemical Localization of an Adenosine Triphosphate Dephosphorylating Enzyme in the Granules of Human Eosinophils

Blood ◽  
1962 ◽  
Vol 19 (5) ◽  
pp. 612-614 ◽  
Author(s):  
DAVID WEINSTEIN ◽  
J. R. SOMMER ◽  
J. W. BEARD
Keyword(s):  
Bone Marrow ◽  
Adenosine Triphosphate ◽  
Myeloid Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Cytochemical Localization ◽  
Normal Human ◽  
Normal Human Bone Marrow

Abstract The granules of the eosinophilic myeloid cells of normal human bone marrow exerted a pronounced activity to dephosphorylate adenosine triphosphate. This behavior was not shown by the granules of other myeloid cells.

scholarly journals The action of bryostatin on normal human hematopoietic progenitors is mediated by accessory cell release of growth factors

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 716-720 ◽  
Author(s):  
SJ Sharkis ◽  
RJ Jones ◽  
ML Bellis ◽  
GD Demetri ◽  
JD Griffin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Single Cell ◽  
Cell Cultures ◽  
Bone Marrow Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Accessory Cell ◽  
Bryostatin 1 ◽  
Gm Csf

Abstract Since enrichment of human bone-marrow hematopoietic progenitors is becoming more feasible and since purified growth factors are now available, we sought to study the action of growth factors on CD34- positive enriched cultures of human bone-marrow cells. We tested the effect of recombinant human (rh) granulocyte-macrophage colony- stimulating factor (GM-CSF), rh interleukin-3 (IL-3), or a unique biologic response modifier, bryostatin 1, on the growth of purified CD34 cells obtained by limiting dilution in single-cell cultures. We have shown previously that bryostatin 1 stimulates both myeloid and erythroid progenitors of human origin in vitro. In this study both IL-3 and GM-CSF supported colony formation from 500, 100, or single-cell cultures at equivalent plating efficiences, suggesting a direct action of these factors on hematopoietic cell growth. Conversely, bryostatin 1 did not support the growth of CD34 cells in single-cell cultures, and the cloning efficiency increased with increasing the number of cells in the culture. To test whether the indirect action of bryostatin 1 might be mediated through the production of growth factors by accessory cells, studies were performed using antibodies directed against human IL-3 and GM-CSF in culture with bryostatin 1 and normal human bone- marrow cells. Results are consistent with the hypothesis that bryostatin 1 could have a stimulatory effect on the accessory cell populations to produce either IL-3 or GM-CSF. Further support for this notion was obtained by demonstrating that T cells, which are cells known to be able to produce IL-3 and GM-CSF, are stimulated by bryostatin 1 to express messenger RNA (mRNA) for specific growth factors, including GM-CSF. These results provide further support that bryostatin 1 may be a useful clinical agent to stimulate hematopoiesis in vivo.

scholarly journals Myofibroblastic stromal cells isolated from human bone marrow induce the proliferation of both early myeloid and B-lymphoid cells

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2396-2405 ◽  
Author(s):  
I Moreau ◽  
V Duvert ◽  
C Caux ◽  
MC Galmiche ◽  
P Charbord ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Myeloid Cells ◽  
Cell Types ◽  
Lymphoid Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Present System ◽  
Normal Human ◽  
B Lymphoid

Abstract Normal human bone marrow stromal cells (BMSC) were isolated from Dexter- type long-term cultures according to their capacity to adhere to plastic and to their lack of hematopoietic antigens. The BMSC displayed a homogeneous appearance and a myofibroblastic phenotype in culture. The stromal cells (SC) were shown to support the proliferation of purified CD34+ hematopoietic progenitors and permitted us to maintain myeloid cells for several weeks in culture. In addition, the BMSC induced the proliferation of purified CD10+ s mu- fetal BM B-cell precursors (BCP). The capacity of the BMSC to induce the proliferation of early myeloid cells was shared by several other human fibroblastic- like cell types. In contrast, the BMSC were far superior to other adherent cells for induction of BCP proliferation. This capacity was largely mediated by endogenously produced interleukin-7 (IL-7), because it could be inhibited by anti-IL-7 antibody. In line with this finding, addition of IL-7 considerably enhanced BCP proliferation in cocultures with skin fibroblasts or synoviocytes. Thus, production of IL-7 appears to be a critical parameter that determines the ability of fibroblastic- like cells to induce BCP proliferation. Taken together, our data show that normal human myofibroblastic BMSC induce the proliferation of both early myeloid and B-lymphoid cells in the absence of accessory hematopoietic cells. The present system should constitute a model to study interactions between native human BM myofibroblastic stroma and various hematopoietic cell subsets.

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