scholarly journals Purification of hemopoietic progenitor cells from human marrow using a fucose-binding lectin and cell sorting

Blood ◽  
1980 ◽  
Vol 56 (5) ◽  
pp. 798-805 ◽  
Author(s):  
G Morstyn ◽  
NA Nicola ◽  
D Metcalf
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Cell Sorting ◽  
Human Peripheral Blood ◽  
Polymorphonuclear Cells ◽  
Fold Enrichment ◽  
Blast Cells ◽  
Binding Lectin ◽  
Selection Of ◽  
Marrow Cells

Abstract Human peripheral blood granulocytes, but not lymphocytes, erythrocytes, or monocytes, bound the fucose-binding lectin from Lotus tetragonolobus (FBP), and this binding was competitively inhibited by the sugar alpha- L-fucose. The fluorescence-activated cell sorter was used to study the appearance of this receptor on human marrow cells during granulocyte differentiation and to prepare fractions enriched for granulocyte- macrophage progenitor cells (granulocyte-macrophage colony-forming cells--GM-CFC). Cell binding of fluoresceinated FBP increased for bone marrow cells in the sequence--lymphocytes, blast cells, promyelocytes and myelocytes, monocytes, and polymorphonuclear cells. Selection of cells with appropriate low-angle or high-angle light scatter characteristics achieved a 10-fold or 2–3-fold enrichment of progenitor cells, respectively. By selecting cells with intermediate fluorescence intensity, a further 2–3-fold enrichment for GM-CFC was obtained. Cell sorting using the optimal selection of these three parameters produced up to 36-fold enrichment of the progenitor cells from human bone marrow. The most enriched fraction was composed of 23% progenitor cells (colony- and cluster-forming cells) with a yield of 36%. In populations most highly enriched by GM-CFC, immature cells (blast cells, promyelocytes, and myelocytes) made up 95% of the cells present.

scholarly journals Purification of hemopoietic progenitor cells from human marrow using a fucose-binding lectin and cell sorting

Blood ◽  
1980 ◽  
Vol 56 (5) ◽  
pp. 798-805
Author(s):  
G Morstyn ◽  
NA Nicola ◽  
D Metcalf
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Cell Sorting ◽  
Human Peripheral Blood ◽  
Polymorphonuclear Cells ◽  
Fold Enrichment ◽  
Blast Cells ◽  
Binding Lectin ◽  
Selection Of ◽  
Marrow Cells

Human peripheral blood granulocytes, but not lymphocytes, erythrocytes, or monocytes, bound the fucose-binding lectin from Lotus tetragonolobus (FBP), and this binding was competitively inhibited by the sugar alpha- L-fucose. The fluorescence-activated cell sorter was used to study the appearance of this receptor on human marrow cells during granulocyte differentiation and to prepare fractions enriched for granulocyte- macrophage progenitor cells (granulocyte-macrophage colony-forming cells--GM-CFC). Cell binding of fluoresceinated FBP increased for bone marrow cells in the sequence--lymphocytes, blast cells, promyelocytes and myelocytes, monocytes, and polymorphonuclear cells. Selection of cells with appropriate low-angle or high-angle light scatter characteristics achieved a 10-fold or 2–3-fold enrichment of progenitor cells, respectively. By selecting cells with intermediate fluorescence intensity, a further 2–3-fold enrichment for GM-CFC was obtained. Cell sorting using the optimal selection of these three parameters produced up to 36-fold enrichment of the progenitor cells from human bone marrow. The most enriched fraction was composed of 23% progenitor cells (colony- and cluster-forming cells) with a yield of 36%. In populations most highly enriched by GM-CFC, immature cells (blast cells, promyelocytes, and myelocytes) made up 95% of the cells present.

scholarly journals Thrombopoietin Is Synthesized by Bone Marrow Stromal Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3444-3455 ◽  
Author(s):  
Anastasia Guerriero ◽  
Lydia Worford ◽  
H. Kent Holland ◽  
Gui-Rong Guo ◽  
Kevin Sheehan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Cell Sorting ◽  
Bone Marrow Cells ◽  
Adult Bone Marrow ◽  
Hla Dr ◽  
Adult Bone ◽  
Marrow Cells

Abstract We have previously characterized stromal progenitor cells contained in fetal bone marrow by fluorescence-activated cell sorting (FACS) using the differential expression of CD34, CD38, and HLA-DR, and found that a small number were contained within the CD34+ cell fraction. In the present study, the frequency of stromal progenitors in both the CD34+ and CD34− subpopulations from samples of fetal and adult bone marrow was approximately one in 5,000 of the mononuclear cell fraction. Using multiparameter single-cell sorting, one in 20 fetal bone marrow cells with the CD34+, CD38−, HLA-DR−, CDw90+ phenotype were clonogenic stromal progenitors, whereas greater than one in five single cells with the CD34−, CD38−, HLA-DR−, CDw90+ phenotype formed stromal cultures. We found that cultures initiated by hematopoietic and stromal progenitors contained within the CD34+ fraction of bone marrow cells formed mixed hematopoietic/stromal cell cultures that maintained the viability of the hematopoietic progenitor cells for 3 weeks in the absence of added hematopoietic cytokines. We characterized some of the hematopoietic cytokines synthesized by stromal cultures derived from either CD34+ or CD34− bone marrow cells using reverse transcriptase–polymerase chain reaction (RT-PCR) amplification of interleukin-3 (IL-3), stem cell factor (SCF), CD34, Flt3/Flk2 ligand (FL), and thrombopoietin (TPO) mRNA sequences. We found ubiquitous expression of TPO mRNA in greater than 90% of stromal cultures initiated by either CD34+ or CD34− cells, and variable expression of SCF, FL, and CD34 mRNA. In particular, SCF and CD34 mRNA were detected only in stromal cultures initiated by CD34+ bone marrow cells, although the differences between CD34+ and CD34− stromal cells were not statistically significant. IL-3 mRNA was not found in any stromal cultures. An enzyme-linked immunosorbent assay (ELISA) of soluble SCF and TPO present in culture supernatants demonstrated that biologically significant amounts of protein were secreted by some cultured stromal cells: eight of 16 samples of conditioned media from stromal cultures initiated by fetal and adult bone marrow contained more than 32 pg/mL SCF (in the linear range of the ELISA), with a median value of 32 pg/mL (range, 9 to 230), while 13 of 24 samples of conditioned media had more than 16 pg/mL TPO (in the linear range of the ELISA), with a median of 37 pg/mL (range, 16 to 106). Our data indicate that stromal cultures initiated by single bone marrow cells can make FL, SCF, and TPO. Local production of early-acting cytokines and TPO by stromal cells may be relevant to the regulation of hematopoietic stem cell self-renewal and megakaryocytopoiesis in the bone marrow microenvironment.

Murine pluripotent hematopoietic progenitors constitutively expressing a normal erythropoietin receptor proliferate in response to erythropoietin without preferential erythroid cell differentiation

1994 ◽  
Vol 14 (7) ◽  
pp. 4834-4842
Author(s):  
A Dubart ◽  
F Feger ◽  
C Lacout ◽  
F Goncalves ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Erythropoietin Receptor ◽  
Erythroid Cell ◽  
Major Influence ◽  
Proliferative Potential ◽  
Steel Factor ◽  
Interleukin 3 ◽  
Marrow Cells

Erythropoietin (EPO) is a prime regulator of the growth and differentiation of erythroid blood cells. The EPO receptor (EPO-R) is expressed in late erythroid progenitors (mature BFU-E and CFU-E), and EPO induces proliferation and differentiation of these cells. By introducing, with a retroviral vector, a normal EPO-R cDNA into murine adult bone marrow cells, we showed that EPO is also able to induce proliferation in pluripotent progenitor cells. After 7 days of coculture with virus-producing cells, bone marrow cells were plated in methylcellulose culture in the presence of EPO, interleukin-3, or Steel factor alone or in combination. In the presence of EPO alone, EPO-R virus-infected bone marrow cells gave rise to mixed colonies comprising erythrocytes, granulocytes, macrophages and megakaryocytes. The addition of interleukin-3 or Steel factor to methylcellulose cultures containing EPO did not significantly modify the number of mixed colonies. The cells which generate these mixed colonies have a high proliferative potential as shown by the size and the ability of the mixed colonies to give rise to secondary colonies. Thus, it appears that EPO has the same effect on EPO-R-expressing multipotent cell proliferation as would a combination of several growth factors. Finally, our results demonstrate that inducing pluripotent progenitor cells to proliferate via the EPO signaling pathway has no major influence on their commitment.

Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 700-704 ◽  
Author(s):  
Kimberly A. Gush ◽  
Kai-Ling Fu ◽  
Markus Grompe ◽  
Christopher E. Walsh
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mitomycin C ◽  
Fanconi Anemia ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Hematopoietic Stem ◽  
Marrow Cells

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc −/− nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity offancc −/− hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc −/− bone marrow cells were transduced with the use of retrovirus carrying the humanfancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment offancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.

Enhanced endothelialization and microvessel formation in polyester grafts seeded with CD34+ bone marrow cells

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 581-585 ◽  
Author(s):  
Vishwanath Bhattacharya ◽  
Peter A. McSweeney ◽  
Qun Shi ◽  
Benedetto Bruno ◽  
Atsushi Ishida ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Vascular Graft ◽  
Bone Marrow Cells ◽  
Venous Blood ◽  
Immunomagnetic Bead ◽  
Composite Grafts ◽  
Cd34 Cells ◽  
Marrow Cells

The authors have shown accelerated endothelialization on polyethylene terephthalate (PET) grafts preclotted with autologous bone marrow. Bone marrow cells have a subset of early progenitor cells that express the CD34 antigen on their surfaces. A recent in vitro study has shown that CD34+ cells can differentiate into endothelial cells. The current study was designed to determine whether CD34+ progenitor cells would enhance vascular graft healing in a canine model. The authors used composite grafts implanted in the dog's descending thoracic aorta (DTA) for 4 weeks. The 8-mm × 12-cm composite grafts had a 4-cm PET graft in the center and 4-cm standard ePTFE grafts at each end. The entire composite was coated with silicone rubber to make it impervious; thus, the PET segment was shielded from perigraft and pannus ingrowth. There were 5 study grafts and 5 control grafts. On the day before surgery, 120 mL bone marrow was aspirated, and CD34+ cells were enriched using an immunomagnetic bead technique, yielding an average of 11.4 ± 5.3 × 106. During surgery, these cells were mixed with venous blood and seeded onto the PET segment of composite study grafts; the control grafts were treated with venous blood only. Hematoxylin and eosin, immunocytochemical, and AgNO3staining demonstrated significant increases of surface endothelialization on the seeded grafts (92% ± 3.4% vs 26.6% ± 7.6%; P = .0001) with markedly increased microvessels in the neointima, graft wall, and external area compared with controls. In dogs, CD34+ cell seeding enhances vascular graft endothelialization; this suggests practical therapeutic applications.

scholarly journals An Efficient Method for Generating Murine Hypothalamic Neurospheres for the Study of Regional Neural Progenitor Biology

Endocrinology ◽  
2020 ◽  
Vol 161 (4) ◽  
Author(s):  
Dinushan Nesan ◽  
Hayley F Thornton ◽  
Laronna C Sewell ◽  
Deborah M Kurrasch
Keyword(s):  
Progenitor Cells ◽  
Sonic Hedgehog ◽  
Cell Sorting ◽  
Seeding Density ◽  
Controlled Environment ◽  
Stem And Progenitor Cells ◽  
A New Technique ◽  
Primary Effector ◽  
Selection Of

Abstract The hypothalamus is a key homeostatic brain region and the primary effector of neuroendocrine signaling. Recent studies show that early embryonic developmental disruption of this region can lead to neuroendocrine conditions later in life, suggesting that hypothalamic progenitors might be sensitive to exogenous challenges. To study the behavior of hypothalamic neural progenitors, we developed a novel dissection methodology to isolate murine hypothalamic neural stem and progenitor cells at the early timepoint of embryonic day 12.5, which coincides with peak hypothalamic neurogenesis. Additionally, we established and optimized a culturing protocol to maintain multipotent hypothalamic neurospheres that are capable of sustained proliferation or differentiation into neurons, oligodendrocytes, and astrocytes. We characterized media requirements, appropriate cell seeding density, and the role of growth factors and sonic hedgehog (Shh) supplementation. Finally, we validated the use of fluorescence activated cell sorting of either Sox2GFPKI or Nkx2.1GFPKI transgenic mice as an alternate cellular isolation approach to enable enriched selection of hypothalamic progenitors for growth into neurospheres. Combined, we present a new technique that yields reliable culturing of hypothalamic neural stem and progenitor cells that can be used to study hypothalamic development in a controlled environment.

scholarly journals The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors

Blood ◽  
1995 ◽  
Vol 85 (9) ◽  
pp. 2422-2435 ◽  
Author(s):  
EK Waller ◽  
J Olweus ◽  
F Lund-Johansen ◽  
S Huang ◽  
M Nguyen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Progenitor Cells ◽  
Cell Sorting ◽  
Single Cells ◽  
Light Scatter ◽  
Hematopoietic Progenitor ◽  
Individual Culture ◽  
Hla Dr ◽  
Growth Properties

There is a long-standing controversy as to whether a single bone marrow (BM)-derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence- activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38- , HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM.

Cell surface peptidase CD26/DPPIV mediates G-CSF mobilization of mouse progenitor cells

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4680-4686 ◽  
Author(s):  
Kent W. Christopherson ◽  
Scott Cooper ◽  
Hal E. Broxmeyer
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mouse Bone Marrow ◽  
Peptidase Activity ◽  
Hematopoietic Stem ◽  
Migratory Response ◽  
Stromal Cell Derived Factor ◽  
Marrow Cells

AbstractCXC ligand 12 (CXCL12; also known as stromal cell–derived factor 1α/SDF-1α) chemoattracts hematopoietic stem and progenitor cells (HSCs/HPCs) and is thought to play a crucial role in the mobilization of HSCs/HPCs from the bone marrow. CD26 (dipeptidylpeptidase IV [DPPIV]) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N-terminus of polypeptide chains. CD26 has the ability to cleave CXCL12 at its position-2 proline. We found by flow cytometry that CD26 is expressed on a subpopulation of normal Sca-1+c-kit+lin— hematopoietic cells isolated from mouse bone marrow, as well as Sca-1+c-kit—lin— cells, and that these cells possess CD26 peptidase activity. To test the functional role of CD26 in CXCL12-mediated normal HSC/HPC migration, chemotaxis assays were performed. The CD26 truncated CXCL12(3-68) showed an inability to induce the migration of sorted Sca-1+c-kit+lin— or Sca-1+c-kit—lin— mouse marrow cells compared with the normal CXCL12. In addition, CXCL12(3-68) acts as an antagonist, resulting in the reduction of migratory response to normal CXCL12. Treatment of Sca-1+c-kit+lin— mouse marrow cells, and myeloid progenitors within this population, or Sca-1+c-kit—lin— cells with a specific CD26 inhibitor, enhanced the migratory response of these cells to CXCL12. Finally, to test for potential in vivo relevance of these in vitro observations, mice were treated with CD26 inhibitors during granulocyte colony-stimulating factor (G-CSF)–induced mobilization. This treatment resulted in a reduction in the number of progenitor cells in the periphery as compared with the G-CSF regimen alone. This suggests that a mechanism of action of G-CSF mobilization involves CD26.

Multilineage Expansion of Lymphoid and Myeloid Cells in Mice Expressing PML-RARα under the Control of the Murine Cathepsin G Locus.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2551-2551
Author(s):  
Geoffrey L. Uy ◽  
Jacqueline E. Payton ◽  
Timothy James Ley
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Cell Function ◽  
Cathepsin G ◽  
Pcr Analysis ◽  
Self Renewal ◽  
Marrow Cells

In one of our laboratory’s models of acute promyelocytic leukemia (APL), the PML-RARα fusion cDNA is knocked into the 5′ UT of the azurophil granule protease, cathepsin G. Nearly all mCG-PML-RARα mice develop lethal leukemia with promyelocytic features following a 150–400 day latent period. We originally chose the cathepsin G gene for the targeting locus because its expression was believed to be restricted to promyelocytes. However, we have now observed high levels of cathepsin G expression with gene expression profiling of leukemic bone marrow samples from patients with AML FAB M1 or M2, i.e. blasts with minimal or no differentiation (M1: n=21, mean raw expression value for cathepsin G=23,885 ± 31,737; M2: n=22, mean=24,088 ± 26,585; M3: n=14, mean 116,029 ± 47,017). To examine whether cathepsin G is normally expressed in murine hematopoietic progenitor cells, we performed gene expression arrays with highly purified Sca-1+/lin− bone marrow cells; cathepsin G mRNA was easily detected in these cells (n=4, mean cathepsin G raw expression value=9,324 ± 5,082; array average normalized to 1,500). We therefore decided to determine whether the early progenitor compartment in mCG-PML-RARα mice was expanded due to unexpected expression of the transgene in KLS (c-kit+, Lin−, Sca-1+) cells; however, no difference was detected in the frequency of marrow-derived KLS cells between mCG-PML-RARα and WT mice that were age, strain, and gender matched (0.074% vs 0.071%, p=0.89). The GMP compartment (Lin−, c-kit+, Sca1−, CD34+, FcRγ+) showed a tendency towards expansion in mCG-PML-RARα mice (0.10% vs 0.032%) but the difference was not significant (p=0.067). To better assess stem cell function in these mice, we performed a competitive repopulation study using marrow derived from Ly 5.2/mCG-PML-RARα mice mixed with Ly5.1/WT marrow cells at various ratios (1:1, 9:1, and 1:9) that were transplanted into genetically compatible hosts. Lineage markers in the peripheral blood were tested at 6, 12, and 24 weeks to assess the contribution of mCG-PML-RARα-derived progenitor and stem cells to the B, T, and myeloid lineages. As expected, we observed a highly reproducible increase in the proportion of Gr-1+ cells derived from mCG-PML-RARα donors at all time points (78.8% ± 11% donor-derived cells at a 1:1 donor: recipient ratio at 6 months, p=0.0006). Surprisingly, we also observed a reproducible increase in B220+/CD19+ cells (66.7% ± 4%, p<0.0001) and CD3+ cells from the mCG-PML-RARα donors (60.4% ± 3% p=0.0010) at 6 months, suggesting that mCG-PML-RARα also confers a growth or survival advantage to B and T cells. To determine whether cathepsin G was expressed in these compartments, we purified CD19+ B and CD3+ T cells by flow cytometry (>99% purity) and did not detect cathepsin G mRNA using a sensitive, knockout-proven qRT-PCR analysis. These data strongly suggest that both the WT cathepsin G gene and the mutant mCG-PML-RARα allele are unexpectedly expressed in the stem cell compartment, with both myeloid and lymphoid progeny of these cells displaying a growth advantage in vivo. However, lymphoid malignancies have not been detected in these mice (n>400), suggesting that PML-RARα is unable to initiate transformation in lymphocytes. Our data imply that the PML-RARα gene is activated in a multipotent compartment in this mouse model, raising the possibility that PML-RARα may not actually ‘reprogram’ progenitor cells to undergo self-renewal, but may rather initiate transformation in pluripotent cells with intrinsic self-renewal capabilities.

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