Human breast epithelium in vitro: The re-expression of structural and functional cellular differentiation in long-term culture

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.

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Effect of donor age on long-term culture of bone marrow in vitro

Mechanisms of Ageing and Development ◽

10.1016/0047-6374(84)90179-9 ◽

1984 ◽

Vol 24 (1) ◽

pp. 119-127 ◽

Cited By ~ 10

Author(s):

D.A. Lipschitz ◽

K.B. Udupa

Keyword(s):

Bone Marrow ◽

Donor Age ◽

Long Term Culture

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Growth and antrum formation of bovine primary follicles in long-term culture in vitro

Reproductive Biology ◽

10.1016/j.repbio.2013.06.003 ◽

2013 ◽

Vol 13 (3) ◽

pp. 221-228 ◽

Cited By ~ 13

Author(s):

Jing Sun ◽

Xiangdong Li

Keyword(s):

Culture In Vitro ◽

Long Term Culture

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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 ◽

10.1182/blood.v90.2.641 ◽

1997 ◽

Vol 90 (2) ◽

pp. 641-650 ◽

Cited By ~ 121

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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Secretion of interferon-tau by bovine embryos in long-term culture: comparison of in vivo derived, in vitro produced, nuclear transfer and demi-embryos

Animal Reproduction Science ◽

10.1016/s0378-4320(99)00015-9 ◽

1999 ◽

Vol 55 (3-4) ◽

pp. 151-162 ◽

Cited By ~ 37

Author(s):

M Stojkovic ◽

M Büttner ◽

V Zakhartchenko ◽

J Riedl ◽

H.-D Reichenbach ◽

...

Keyword(s):

Nuclear Transfer ◽

Bovine Embryos ◽

Interferon Tau ◽

Long Term Culture

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Long-term culture of primary B cells and in vitro expression of an exogenous gene

Immunology Letters ◽

10.1016/0165-2478(95)00092-7 ◽

1995 ◽

Vol 47 (3) ◽

pp. 193-197 ◽

Cited By ~ 2

Author(s):

Abhay Kumar ◽

Emily C. Guido ◽

Ru-Shya Liu ◽

Mohammad S. Saedi

Keyword(s):

B Cells ◽

In Vitro Expression ◽

Long Term Culture ◽

Exogenous Gene

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EXPANSION AND LONG-TERM CULTURE OF DIFFERENTIATED NORMAL RAT UROTHELIAL CELLS IN VITRO

In Vitro Cellular & Developmental Biology - Animal ◽

10.1290/1071-2690(2001)037<0419:ealtco>2.0.co;2 ◽

2001 ◽

Vol 37 (7) ◽

pp. 419 ◽

Cited By ~ 24

Author(s):

YUAN YUAN ZHANG ◽

BARBARA LUDWIKOWSKI ◽

ROBERT HURST ◽

PETER FREY

Keyword(s):

Urothelial Cells ◽

Long Term Culture ◽

Normal Rat

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In Vitro Co-Culture of CLL-B Cells Reveals Long-Term Survival, Proliferation, and Maintenance of Telomere Length

Blood ◽

10.1182/blood.v128.22.350.350 ◽

2016 ◽

Vol 128 (22) ◽

pp. 350-350

Author(s):

Ceri H Jones ◽

Thet Thet Lin ◽

Elisabeth Jane Walsby ◽

Guy E Pratt ◽

Christopher Fegan ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Telomere Length ◽

Erosion Rate ◽

Ex Vivo ◽

Effector Memory ◽

Long Term Culture

Abstract Telomere length is a prognostic factor in Chronic Lymphocytic Leukemia (CLL) with short telomere length a powerful predictor of early time to first treatment and reduced overall survival. However, little is known about telomere dynamics through the course of an individual patient's disease. Our recent longitudinal analysis of CLL B-cell telomere length revealed very little dynamic change within individual patients with a mean erosion rate of -52bp/year (p=0.05). In marked contrast, T-cells derived from the same patients showed a significantly higher mean erosion rate of -119bp/year (p=0.02) with a median follow up time of 69 months. Here we present data derived from long-term in-vitro co-culture of peripheral blood from CLL patients coupled with temporal analysis of their telomere length dynamics. We utilized a multi-cellular co-culture system, comprised of autologous T-cells and CD40L-expressing mouse fibroblasts, to maintain CLL cells in long-term culture. Patient-derived peripheral blood mononuclear cells (n=16) were maintained for a median of 70 days (range 54-154); samples were analyzed every two weeks for tumor cell telomere length and evidence of proliferation. We used fluorescence-activated cell sorting (FACS) to sort populations of CD19+CD5+ CLL B-cells and CD3+ T-cells from each of the cultures. We then performed high-resolution single telomere length analysis (STELA) on these sorted subsets of cells and analyzed their telomere dynamics over this extended time course. Analysis of CLL B-cells from these cultures revealed significantly increased Ki-67+ at day 14 when compared to day 0 (p<0.001) and this was evident for the duration of the cultures. Despite sustained tumor cell proliferation, we observed no significant difference in the CLL B-cell telomere length with a mean TL at the start of 4.5kb vs 4.3kb at the end (p=0.14). The presence of T-cells was shown to be critical for the maintenance of the long-term cultures in two ways. Firstly, cultures that were treated with 4μM fludarabine showed a catastrophic reduction in T-cells (p=0.01), which was associated with a significantly shorter duration of survival of CLL B-cells when compared to untreated controls (median 17.5 days (range 7-70); p<0.001). Secondly, it proved impossible to maintain T-cell depleted, purified CLL B-cells, in long-term culture. T-cells isolated from the long-term cultures showed evidence of proliferation with Ki-67+ again being increased at day 14 in comparison to baseline (p=0.003). Furthermore, T-cells derived from these cultures showed a significant alteration in subset composition over time with a decrease in the numbers of naive CD4+ (p=0.05) and CD8+ (p=0.02) T-cells and a corresponding increase in effector memory (p=0.2) and terminally differentiated effector memory (EMRA) subsets (p=0.07). In conclusion, this study demonstrates that we have developed a robust, long-term culture method for the maintenance of CLL cells. Despite evidence of sustained CLL proliferation, CLL B-cells showed little telomere length erosion during long-term co-culture and this is compatible with our recent ex-vivo analysis, which showed that the telomere length of CLL B-cells are remarkably stable with a mean erosion rate of only -52bp/year. In both ex-vivo and in-vitro analysis, telomere erosion correlated with starting telomere length (r2=0.14, p=0.04 and r2=0.3 p=0.03 respectively). Taken together, our in-vitro and ex-vivo data imply that the radically short telomeres observed in some CLL patients are not the result of increased proliferation of the malignant B-cell, but rather the mutagenic event occurs in a B-cell which already has short telomeres. Furthermore, our novel long-term culture model has reinforced the vital role of T-cells in sustaining CLL B-cells viability and proliferation in-vitro. Given the consistent skewing of the T-cell pool towards a memory phenotype it seems unlikely that this is driven in-vitro by cognate TCR antigen recognition but rather a cytokine-mediated response. Disclosures Fegan: Gilead Sciences: Honoraria; Roche: Honoraria; AbbVie: Honoraria.

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