Long-Term Culture of Nephron Progenitor Cells Ex Vivo

2019 ◽  
pp. 63-75
Author(s):  
Aaron C. Brown ◽  
Ashwani K. Gupta ◽  
Leif Oxburgh
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Long Term Culture ◽  
Nephron Progenitor

scholarly journals DNA Methylation Changes duringIn VitroPropagation of Human Mesenchymal Stem Cells: Implications for Their Genomic Stability?

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Angela Bentivegna ◽  
Mariarosaria Miloso ◽  
Gabriele Riva ◽  
Dana Foudah ◽  
Valentina Butta ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Genomic Stability ◽  
Great Promise ◽  
Human Mscs ◽  
Low Oxygen ◽  
Long Term Culture ◽  
Functional Changes

Mesenchymal stem cells (MSCs) hold great promise for the treatment of numerous diseases. A major problem for MSC therapeutic use is represented by the very low amount of MSCs which can be isolated from different tissues; thusex vivoexpansion is indispensable. Long-term culture, however, is associated with extensive morphological and functional changes of MSCs. In addition, the concern that they may accumulate stochastic mutations which lead the risk of malignant transformation still remains. Overall, the genome of human MSCs (hMSCs) appears to be apparently stable throughout culture, though transient clonal aneuploidies have been detected. Particular attention should be given to the use of low-oxygen environment in order to increase the proliferative capacity of hMSCs, since data on the effect of hypoxic culture conditions on genomic stability are few and contradictory. Furthermore, specific and reproducible epigenetic changes were acquired by hMSCs duringex vivoexpansion, which may be connected and trigger all the biological changes observed. In this review we address current issues on long-term culture of hMSCs with a 360-degree view, starting from the genomic profiles and back, looking for an epigenetic interpretation of their genetic stability.

Stable Phenotype and Function of Immortalized Lin−Sca-1+ Cardiac Progenitor Cells in Long-Term Culture: A Step Closer to Standardization

2014 ◽  
Vol 23 (9) ◽  
pp. 1012-1026 ◽  
Author(s):  
Ana G. Freire ◽  
Diana S. Nascimento ◽  
Giancarlo Forte ◽  
Mariana Valente ◽  
Tatiana P. Resende ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cardiac Progenitor Cells ◽  
Long Term Culture ◽  
And Function

Long-term culture of murine erythropoietic progenitor cells in the absence of an adherent layer

Nature ◽  
1983 ◽  
Vol 305 (5935) ◽  
pp. 625-627 ◽  
Author(s):  
Françoise Wendling ◽  
Mona M. Shreeve ◽  
David L. McLeod ◽  
Arthur A. Axelrad
Keyword(s):  
Progenitor Cells ◽  
Long Term Culture

Ex-Vivo Treatment of Graft by Long-Term Culture

1993 ◽  
pp. 250-254
Author(s):  
J. Chang ◽  
T. M. Dexter
Keyword(s):  
Ex Vivo ◽  
Long Term Culture

scholarly journals Maintenance of transplantation potential in ex vivo expanded CD34(+)- selected human peripheral blood progenitor cells

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2898-2903 ◽  
Author(s):  
R Henschler ◽  
W Brugger ◽  
T Luft ◽  
T Frey ◽  
R Mertelsmann ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Large Scale ◽  
Ex Vivo ◽  
Human Peripheral Blood ◽  
Ex Vivo Expansion ◽  
High Dose ◽  
Peripheral Blood Progenitor Cells ◽  
Limiting Dilution

Abstract CD34(+)-selected hematopoietic progenitor cells are being increasingly used for autotransplantation, and recent evidence indicates that these cells can be expanded ex vivo. Of 15 patients with solid tumors undergoing a phase I/II clinical trial using CD34(+)-selected peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy, we analyzed the frequency of long-term culture-initiating cells (LTCIC) as a measure of transplantation potential before and after ex vivo expansion of CD34+ cells. PBPCs were mobilized by combination chemotherapy and granulocyte colony-stimulating factor (G-CSF). The original unseparated leukapheresis preparations, the CD34(+)-enriched transplants, as well as nonabsorbed fractions eluting from the CD34 immunoaffinity columns (Ceprate; CellPro, Bothell, WA) were monitored for their capacity to repopulate irradiated allogeneic stroma in human long-term bone marrow cultures. We found preservation of more than three quarters of fully functional LTCIC in the CD34(+)-selected fractions. Quantitation of LTCIC by limiting dilution analysis showed a 53-fold enrichment of LTCIC from 1/9,075 in the unseparated cells to an incidence of 1/169 in the CD34+ fractions. Thus, in a single apheresis, it was possible to harvest a median of 1.65 x 10(4) LTCIC per kg body weight (range, 0.71 to 3.72). In addition, in six patients, large-scale ex vivo expansions were performed using a five-factor cytokine combination consisting of stem cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (EPO), previously shown to expand committed progenitor cells. LTCIC were preserved, but not expanded during the culture period. Optimization of ex vivo expansion growth factor requirements using limiting dilution assays for LTCIC estimation indicated that the five-factor combination using SCF, IL-1, IL-3, IL-6, and EPO together with autologous plasma was the most reliable combination securing both high progenitor yield and, at the same time, optimal preservation of LTCIC. Our data suggest that ex vivo-expanded CD34+ PBPCs might be able to allow long-term reconstitution of hematopoiesis.

Adhesion to Fibronectin Maintains Regenerative Capacity During Ex Vivo Culture and Transduction of Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4612-4621 ◽  
Author(s):  
M.A. Dao ◽  
K. Hashino ◽  
I. Kato ◽  
J.A. Nolta
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Retroviral Vectors ◽  
Current Data ◽  
Regenerative Capacity ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Ex Vivo Culture

Abstract Recent reports have indicated that there is poor engraftment from hematopoietic stem cells (HSC) that have traversed cell cycle ex vivo. However, inducing cells to cycle in culture is critical to the fields of ex vivo stem cell expansion and retroviral-mediated gene therapy. Through the use of a xenograft model, the current data shows that human hematopoietic stem and progenitor cells can traverse M phase ex vivo, integrate retroviral vectors, engraft, and sustain long-term hematopoiesis only if they have had the opportunity to engage their integrin receptors to fibronectin during the culture period. If cultured in suspension under the same conditions, transduction is undetectable and the long-term multilineage regenerative capacity of the primitive cells is severely diminished.

scholarly journals Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3229-3236 ◽  
Author(s):  
P Gupta ◽  
JB McCarthy ◽  
CM Verfaillie
Keyword(s):  
Stem Cell ◽  
Heparan Sulfate ◽  
Ex Vivo ◽  
Function Analysis ◽  
Soluble Form ◽  
Stromal Fibroblast ◽  
Colony Stimulating Factor ◽  
Long Term Culture ◽  
Stimulating Factor

We have recently demonstrated that 50% of primitive human long-term culture-initiating cells (LTC-IC) are maintained for up to 8 weeks in stroma-dependent cultures in which progenitor-stroma contact is prevented (stroma noncontact), or when progenitors are cultured in medium conditioned by stromal feeders. This indicates that factors responsible for LTC-IC maintenance are present in soluble form in stromal supernatant (SN). Although the picogram concentrations of cytokines present in stromal SN can induce the differentiation of CD34+/HLA-DR- (DR-) cells to clonogenic cells (colony forming cells; CFC), they maintain only 10% of LTC-IC for 5 weeks, suggesting that factors other than these cytokines are required for LTC-IC maintenance. To characterize the factor(s) in stromal SN responsible for LTC-IC maintenance, we purified glycoproteins and proteoglycans (PG) from the SN of the LTC-IC supportive murine marrow stromal fibroblast cell line M2–10B4 by ion exchange high performance liquid chromatography (HPLC). Culture of DR- cells in a combination of M2–10B4-derived PG, but not glycoproteins and picogram concentrations of recombinant human interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), granulocyte- macrophage colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1alpha (MIP-1alpha) resulted in the recovery of 96% +/- 8% of LTC-IC maintained in cultures supplemented with unfractionated stromal SN. LTC-IC maintenance was largely retained after digestion of the PG-rich fraction with proteinase K and after dissociative gel filtration chromatography, but was completely abolished following treatment with nitrous acid, which digests heparan sulfate glycosaminoglycans (HS GAG). As for M2–10B4-derived HS GAG, high concentrations of bovine kidney HS GAG, but not bovine tracheal chondroitin sulfate, significantly improved cytokine-mediated LTC-IC maintenance. Maintenance of LTC-IC by these nonmarrow-derived HS GAG was, however, significantly lower than that seen with M2–10B4-derived HS. These studies demonstrate a role for marrow stroma-derived HS GAG in the long-term in vitro maintenance of human LTC-IC. Further structure-function analysis of these HS GAG may have important implications for ex vivo stem cell expansion and gene transfer into hematopoietic progenitors.

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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