Modified in vitro conditions for cord blood–derived long-term culture-initiating cells

2001 ◽  
Vol 29 (3) ◽  
pp. 309-314 ◽  
Author(s):  
Marina Podestà ◽  
Giovanna Piaggio ◽  
Anna Pitto ◽  
Elena Zocchi ◽  
Monica Soracco ◽  
...  
Keyword(s):  
Cord Blood ◽  
Long Term Culture

scholarly journals Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progenitor population

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3306-3313 ◽  
Author(s):  
QL Hao ◽  
FT Thiemann ◽  
D Petersen ◽  
EM Smogorzewska ◽  
GM Crooks
Keyword(s):  
Cord Blood ◽  
Hematopoietic Progenitors ◽  
Generative Capacity ◽  
Long Term Culture ◽  
Clonal Proliferation ◽  
Unit Cells ◽  
Functional Hierarchy

Long-term culture-initiating cells (LTC-IC) are hematopoietic progenitors able to generate colony-forming unit-cells (CFU) after 5 to 8 weeks (35 to 60 days) of culture on bone marrow (BM) stroma and represent the most primitive progenitors currently detectable in vitro. We have recently reported that long-term cultures initiated with CD34+CD38- cells from BM or cord blood are able to continue generating CFU for at least 100 days, ie, beyond the standard LTC-IC period. In this report, single-cell cultures from cord blood and retroviral marking of cord blood and BM were used to study whether the subpopulation of CD34+CD38- cells able to generate CFU beyond 60 days (“extended long-term culture-initiating cells” or ELTC-IC) are functionally distinct from LTC-IC in terms of timing of initial clonal proliferation and generative capacity. All cord blood LTC-IC formed clones of greater than 50 cells by day 30. In contrast, cord blood ELTC-IC proliferated later in culture, 50% forming clones after day 30. Although efficient retroviral marking of LTC-IC was seen (25% to 45%), marking of ELTC-IC was inefficient (< 1%), consistent with a more quiescent progenitor population. There was a positive correlation between time of clonal proliferation and generative capacity. ELTC-IC generated threefold to fourfold more progeny than did LTC-IC (P < .002). These studies show that there is a functional hierarchy of progenitors in long-term culture which correlates with their level of quiescence. By extending the LTC-IC assay, a more primitive progenitor may be studied that may be functionally closer to the human long-term repopulation stem cell in vivo.

scholarly journals AC133+G0Cells from Cord Blood Show a High Incidence of Long-Term Culture-Initiating Cells and a Capacity for More Than 100 Million-Fold Amplification of Colony-Forming Cells In Vitro

Stem Cells ◽  
2004 ◽  
Vol 22 (5) ◽  
pp. 704-715 ◽  
Author(s):  
Yvonne J. Summers ◽  
Clare M. Heyworth ◽  
Erika A. de Wynter ◽  
Claire A. Hart ◽  
James Chang ◽  
...  
Keyword(s):  
Cord Blood ◽  
Long Term Culture ◽  
High Incidence

Low concentrations of suramin can reduce in vitro infection of human cord blood lymphocytes with HTLV-I during long-term culture

1987 ◽  
Vol 8 (5-6) ◽  
pp. 247-260 ◽  
Author(s):  
Caterina D. Pesce ◽  
Fabrizio Ciprani ◽  
Chiara D'Onofrio ◽  
Ester Alvino ◽  
Carlo F. Perno ◽  
...  
Keyword(s):  
Cord Blood ◽  
Human Cord Blood ◽  
Long Term Culture ◽  
Blood Lymphocytes ◽  
Low Concentrations ◽  
Cord Blood Lymphocytes

scholarly journals Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4773-4777 ◽  
Author(s):  
Hal E. Broxmeyer ◽  
Man-Ryul Lee ◽  
Giao Hangoc ◽  
Scott Cooper ◽  
Nutan Prasain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

scholarly journals Loss of primitive hematopoietic progenitors in patients with human immunodeficiency virus infection

Blood ◽  
1996 ◽  
Vol 88 (12) ◽  
pp. 4568-4578 ◽  
Author(s):  
A Marandin ◽  
A Katz ◽  
E Oksenhendler ◽  
M Tulliez ◽  
F Picard ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Human Immunodeficiency Virus ◽  
Long Term Culture ◽  
Cd34 Cells ◽  
Immunodeficiency Virus ◽  
Cell Fractions ◽  
Cd38 Antigen ◽  
Hiv 1

A number of hematologic abnormalities, including cytopenias, have been observed in patients with human immunodeficiency virus (HIV) infection. To elucidate their mechanisms, primitive cells from bone marrow aspirates of 21 patients with HIV-1 infection were quantitated by flow cytometry. The mean percentage of CD34+ cells is not significantly altered in HIV-1-infected patients in comparison with HIV-1- seronegative controls. In contrast, two- and three-color immunofluorescence analysis showed that in all HIV-1 samples, most CD34+ cells coexpressed the CD38 antigen. The proportion of HIV-1- derived CD34+ cells that did not express the CD38 antigen was significantly lower (HIV-1+: mean, 1.73%; controls: mean, 14%; P < .0005) than in controls. Moreover, of Thy-1+ cells, the proportion of CD34+ cells was twofold lower in HIV-1-infected patients (HIV-1+: mean, 12%; controls, 25%, P < .0005), which suggests that phenotypically primitive cells are depleted in HIV-1 infection. In vitro functional analysis in long-term cultures of sorted CD34+ cells from seven HIV-1 patients showed that CD34+ cells from HIV-1 patients generated much fewer colonies both in the nonadherent and adherent layers than CD34+ cells from controls after 5 weeks of culture (10-fold and four-fold less, respectively). Precise long-term culture initiating cell (LTC-IC) frequency in the CD34+ cell population was determined in three patients by limiting dilution and was markedly decreased in comparison to that of normal controls (from twofold to > sevenfold decreased). To determine if primitive cells were infected by HIV-1, both methylcellulose colonies generated from long-term culture of CD34+ cells and various CD34+ cell fractions purified by flow cytometry were evaluated for the presence of HIV-1 by polymerase chain reaction (PCR). Progeny from long-term culture was HIV-1-negative in three samples. In addition, using a sensitive PCR technique, the HIV-1 genome could not be detected in CD34+, CD34+/CD38-, and CD34+/CD4+ cells. These data show that hematologic disorders in HIV disease may be the consequence of a deficit of primitive cells. However, direct infection of these cells by HIV-1 does not seem to be responsible for this defect.

scholarly journals Differential Maintenance of Primitive Human SCID-Repopulating Cells, Clonogenic Progenitors, and Long-Term Culture-Initiating Cells After Incubation on Human Bone Marrow Stromal Cells

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 641-650 ◽  
Author(s):  
Olga I. Gan ◽  
Barbara Murdoch ◽  
Andre Larochelle ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Stromal Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Long Term Culture ◽  
Cell Engraftment ◽  
Ex Vivo Culture

Abstract Many experimental and clinical protocols are being developed that involve ex vivo culture of human hematopoietic cells on stroma or in the presence of cytokines. However, the effect of these manipulations on primitive hematopoietic cells is not known. Our severe combined immune-deficient mouse (SCID)-repopulating cell (SRC) assay detects primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of immune-deficient non-obese diabetic/SCID (NOD/SCID) mice. We have examined here the maintenance of SRC, colony-forming cells (CFC), and long-term culture-initiating cells (LTC-IC) during coculture of adult human BM or umbilical cord blood (CB) cells with allogeneic human stroma. Transplantation of cultured cells in equivalent doses as fresh cells resulted in lower levels of human cell engraftment after 1 and 2 weeks of culture for BM and CB, respectively. Similar results were obtained using CD34+-enriched CB cells. By limiting dilution analysis, the frequency of SRC in BM declined sixfold after 1 week of culture. In contrast to the loss of SRC as measured by reduced repopulating capacity, the transplanted inocula of cultured cells frequently contained equal or higher numbers of CFC and LTC-IC compared with the inocula of fresh cells. The differential maintenance of CFC/LTC-IC and SRC suggests that SRC are biologically distinct from the majority of these in vitro progenitors. This report demonstrates the importance of the SRC assay in the development of ex vivo conditions that will allow maintenance of primitive human hematopoietic cells with repopulating capacity.

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