Ex-vivo expansion of bone marrow progenitor cells for hematopoietic reconstitution following high-dose chemotherapy for breast cancer

Bachier, Carlos; Gökmen, Erhan; Teale, Judy M.; Lanzkron, Sophie; Childs, Craig C.; Franklin, Wilbur A.; Shpall, Elizabeth J.; Douville, Judith; Weber, Stephanie; Müller, Thomas; Armstrong, Douglas; Lemaistre, Charles F.

doi:10.1016/s0301-472x(98)00085-x

Cited by 49 publications

(24 citation statements)

References 32 publications

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“…The in vitro expansion of hematopoietic stem cells is a very promising approach for different clinical applications, ranging from rescue after myeloablative therapy to purging of contaminating tumor cells or other graft engineering techniques [17,18]. The major goals of this study were to define the optimal culture conditions for the in vitro expansion of Lin -stem cells in a short-term, stroma-free, static culture and to provide new insights into the procedures that should be used in a clinical setting.…”

Section: Discussionmentioning

confidence: 99%

In vitro expansion of Lin+ and Lin− mononuclear cells from human peripheral blood

Norhaiza

Rohaya

Zarina

et al. 2013

AIP Conference Proceedings

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Abstract. Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin -) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin + ) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin -cell population. The ability of Lin + and Lin -to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin + and Lin -were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin + mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin -stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin -stem cells for at least 18 days. The Lin -stem cells started to response to the cytokines added as early as Day 2 of culture. It is concluded that Lin -stem cells can be expanded in vitro by culturing in proliferation medium supplemented with cytokines.

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Section: Discussionmentioning

confidence: 99%

In vitro expansion of Lin+ and Lin− mononuclear cells from human peripheral blood

Norhaiza

Rohaya

Zarina

et al. 2013

AIP Conference Proceedings

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“…The ex vivo expansion of hematopoietic stem cells is a very promising approach for different clinical applications, ranging from rescue after myeloablative therapy to other graft engineering techniques [15,16]. Self-renewal, proliferation, and differentiation of hematopoietic stem cells are regulated by a complex mechanism, which involves the bone marrow microenvironment, where stimulating and inhibiting cytokines as well as cell-to-cell and cell-to-extracellular matrix interactions play a pivotal role.…”

Section: Discussionmentioning

confidence: 99%

The comparison of different protocols for expansion of umbilical‐cord blood hematopoietic stem cells

Chivu¹,

Diaconu²,

Bleoţu³

et al. 2004

J Cellular Molecular Medi

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Hematopoiesis is maintained by the activity of multipotent stem cells, which have the dual capacity to self‐renew and to differentiate into all of the blood cell lineages. The major challenge of stem cells based regenerative therapy is to expand ex vivo the primitive compartment to increase transplantable stem cells number. The present study was designed to evaluate several culture systems for in vitro maintenance of umbilical cord blood stem cells. The influences of different growth conditions such as stromal feeder layer, cytokines supplement and placental conditioned medium (PCM) have been evaluated over a relatively short period of time on CD34+ cell expansion and maintenance of clonogenic progenitors. When cells were expanded on feeder layer in the presence of added cytokines and PCM on average a 2.96‐fold increase of CD34+CD71‐ and a 3.13‐fold increase of CD34+HLA‐DR‐ was observed. The total number of colony forming cells (35±2.65) indicated also that the yield of clonogenic progenitors obtained with a combination of all factors was two folds higher than each of these factors alone and ten time above control (3.67± 2.52). In conclusion, the results of our study clearly show that the ex vivo expansion of hematopoietic progenitor cells obtained from human umbilical cord blood is dependent on controlled experimental conditions, which might be helpful when designing culture systems for clinical applications.

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“…The BM mononuclear cell (MNC) expansion protocol has been previously described. 3 Briefly, approximately 9 × 10 8 BM MNCs purified under Ficoll-Hypaque density gradient were inoculated into the Aastrom's continuous perfusion stromal-coated bioreactors and continuously perfused for 12 days with Iscove's modified Dulbecco's media supplemented with 10% fetal bovine serum, 10% horse serum, hydrocortisone, PIXY321, glutamine, erythropoietin, Flt3-L, gentamicin and vancomycin. The mean fold expansion of MNCs and CFU-GMs were …”

Section: Patients and Control Subjectsmentioning

confidence: 99%

“…Early studies showed the feasibility of using these grafts in clinical trials with regard to myeloid reconstitution and tolerability. [2][3][4] However, proof of multilineage engraftment including lymphopoiesis is needed before ex vivo expanded grafts can be widely used in clinical trials.…”

mentioning

confidence: 99%

Ig heavy chain CDR3 size diversities are similar after conventional peripheral blood and ex vivo expanded hematopoietic cell transplants

Gökmen

Bachier²,

Raaphorst³

et al. 2001

Bone Marrow Transplant

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Summary:It is largely unknown whether the immune repertoire can be reconstituted successfully after high-dose chemotherapy and transplantation using ex vivo expanded hematopoietic stem cell (HSC) grafts. It is critically important for the transplant outcome that immune repertoire reconstitution progresses after ex vivo expanded HSC graft transplants at least as efficiently as that seen after conventional HSC transplants. Previously, we showed that the T cell receptor V beta (TCRVB) third complementarity determining region (CDR3) diversification after ex vivo expanded bone marrow (BM) HSC graft transplants was similar to that seen after conventional peripheral blood stem cell transplants (PBSCTs). In the present study, the CDR3 diversity of the six immunoglobulin (Ig) heavy chain variable region gene (V H ) families was examined in five breast cancer patients who were transplanted with ex vivo expanded BM HSCs as the only source of stem cells. For comparison, 12 healthy adults and four conventional PBSCT recipients were also studied. Using both CDR3 fingerprinting and single strand conformation polymorphism (SSCP) methodologies, it is shown that the contribution of the V H families to the overall repertoire among healthy adults is highly variable and not always proportional to V H family member size. After both ex vivo expanded HSC transplants and conventional PBSCTs, the V H CDR3 repertoires were limited in size diversity at 6 weeks post transplant. By 6 months, however, V H families displayed a repertoire diversity that was as complex as that seen in healthy adults. No difference was seen between ex vivo expanded HSC graft transplant recipients and conventional PBSCT recipients in V H repertoire diversity. In one patient there was a follow-up analysis 12 months after ex vivo expanded graft transplant, and the diversity of the V H families was maintained. In all patients, the amino acid size of the CDR3 regions fell within adult limits at all time points post transplant. These results indicate that B cell reper-

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Ex-vivo expansion of bone marrow progenitor cells for hematopoietic reconstitution following high-dose chemotherapy for breast cancer

Cited by 49 publications

References 32 publications

In vitro expansion of Lin+ and Lin− mononuclear cells from human peripheral blood

In vitro expansion of Lin+ and Lin− mononuclear cells from human peripheral blood

The comparison of different protocols for expansion of umbilical‐cord blood hematopoietic stem cells

Ig heavy chain CDR3 size diversities are similar after conventional peripheral blood and ex vivo expanded hematopoietic cell transplants

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