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

The chronic wound induced by diabetes has poor efficacy and could lead to amputation. The repair function of mesenchymal stem cells (MSCs) impaired after long-term culture in vitro. Studies have shown that the proto-oncogene c-Casitas b-lineage lymphoma (c-Cbl) can regulate receptor- and non-receptor tyrosine kinase, which was also involved in the angiogenesis process. This study aimed to explore the regulative effect of c-Cbl on the proangiogenic functions of long-term cultured MSCs and evaluate its pro-healing effect on diabetic wounds. In this study, the c-Cbl level was downregulated by locked nucleic acid–modified antisense oligonucleotide gapmers (LNA Gapmers). We detected the effect of c-Cbl downregulation on long-term cultured MSCs in terms of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signal, cellular proliferation, senescence, migration, and angiogenic factors paracrine activity in vitro. In vivo, we observed the pro-healing effect of long-term cultured MSCs, with or without c-Cbl downregulation, on the diabetic wound. We found that the phosphorylation level of c-Cbl increased and that of Akt decreased in passage 10 (P10) MSCs compared with passage 3 (P3) MSCs ( P < 0.05). Additionally, the proliferation, paracrine, and migration capacity of P10 MSCs decreased significantly, accompanied by the increase of cellular senescence ( P < 0.05). However, these functions, including PI3K/Akt activity of P10 MSCs, have been improved by c-Cbl downregulation ( P < 0.05). Compared with P10 MSCs treatment, treatment with c-Cbl downregulated P10 MSCs accelerated diabetic wound healing, as defined by a more rapid wound closure ( P < 0.05), more neovascularization ( P < 0.05), and higher scores of wound histological assessment ( P < 0.05) in a diabetic rat model. Our findings suggested that c-Cbl downregulation could attenuate the impairment of proangiogenic functions in MSCs induced by long-term culture in vitro and improve the effect of long-term cultured MSCs in promoting diabetic wound healing.

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

Blood ◽

10.1182/blood.v88.12.4568.bloodjournal88124568 ◽

1996 ◽

Vol 88 (12) ◽

pp. 4568-4578 ◽

Cited By ~ 60

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.

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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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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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