MEK1 activation enhances the ex vivo proliferation of haematopoietic stem/progenitor cell

Abstract We evaluated different culture conditions to obtain a lineage-selected proliferation of clonogenic megakaryocytic progenitors (MP). In low-density (LD) or CD34+ cell cultures, the best results were obtained in serum-free medium in the presence of megakaryocyte growth and development factor, stem cell factor, interleukin-3 (IL-3), IL-6, IL-11, FLT-ligand, and macrophage inflammatory protein-1α. In paired studies, expansion of LD cells was less effective than expansion of CD34+ cells, and pre-enrichment of CD34+ cells using negative depletion of lineage-positive cells produced significantly larger quantities of MP than pre-enrichment using positive selection. MP proliferation peaked on day 7 in culture, and an 8- ± 5-fold expansion of CD34+/CD61+ cells, a 17- ± 5-fold expansion of colony-forming units-megakaryocytes, and a 58- ± 14-fold expansion of the total number of CD61+ cells was obtained. In a feasibility clinical study, 10 cancer patients (8 with breast cancer and 2 with non-Hodgkin's lymphoma) undergoing autologous peripheral blood progenitor cell (PBPC) transplant received MP generated ex vivo (range, 1 to 21 × 105/kg CD61+ cells) together with unmanipulated PBPC. Eight patients received a single allogeneic platelet transfusion, whereas platelet transfusion support was not needed in 2 of the 4 patients receiving the highest doses of cultured MP. This result compares favorably with a retrospective control group of 14 patients, all requiring platelet transfusion support. Adverse reactions or bacterial contamination of cell cultures have not been observed. In conclusion, MP can be expanded ex vivo and safely administered to autologous transplant recipients. Further clinical trials will indicate the reinfusion schedule able to consistently abrogate the need for allogeneic platelet transfusion support in autologous transplantation.

Download Full-text

Ex Vivo Graft Purging and Expansion of Autologous Blood Progenitor Cell Products from Patients with Multiple Myeloma

Cancer Research ◽

10.1158/0008-5472.can-11-0842 ◽

2011 ◽

Vol 71 (14) ◽

pp. 5040-5049 ◽

Cited By ~ 9

Author(s):

Hong Yang ◽

Simon N. Robinson ◽

Yago Nieto ◽

Richard J. Jones ◽

Christopher D. Gocke ◽

...

Keyword(s):

Multiple Myeloma ◽

Progenitor Cell ◽

Ex Vivo ◽

Autologous Blood

Download Full-text

During EPO or anemia challenge, erythroid progenitor cells transit through a selectively expandable proerythroblast pool

Blood ◽

10.1182/blood-2009-12-258947 ◽

2010 ◽

Vol 116 (24) ◽

pp. 5334-5346 ◽

Cited By ~ 17

Author(s):

Arvind Dev ◽

Jing Fang ◽

Pradeep Sathyanarayana ◽

Anamika Pradeep ◽

Christine Emerson ◽

...

Keyword(s):

Stromal Cells ◽

Progenitor Cell ◽

Ex Vivo ◽

Erythropoietin Receptor ◽

Erythroid Cell ◽

Short Term ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Stage Development ◽

Bcl2 Expression

Abstract Investigations of bone marrow (BM) erythroblast development are important for clinical concerns but are hindered by progenitor cell and tissue availability. We therefore sought to more specifically define dynamics, and key regulators, of the formation of developing BM erythroid cell cohorts. A unique Kit−CD71highTer119− “stage E2” proerythroblast pool first is described, which (unlike its Kit+ “stage E1” progenitors, or maturing Ter119+ “stage E3” progeny) proved to selectively expand ∼ 7-fold on erythropoietin challenge. During short-term BM transplantation, stage E2 proerythroblasts additionally proved to be a predominantly expanded progenitor pool within spleen. This E1→E2→E3 erythroid series reproducibly formed ex vivo, enabling further characterizations. Expansion, in part, involved E1 cell hyperproliferation together with rapid E2 conversion plus E2 stage restricted BCL2 expression. Possible erythropoietin/erythropoietin receptor proerythroblast stage specific events were further investigated in mice expressing minimal erythropoietin receptor alleles. For a hypomorphic erythropoietin receptor-HM allele, major defects in erythroblast development occurred selectively at stage E2. In addition, stage E2 cells proved to interact productively with primary BM stromal cells in ways that enhanced both survival and late-stage development. Overall, findings reveal a novel transitional proerythroblast compartment that deploys unique expansion devices.

Download Full-text

Memory Effector Cells but Not Effector Cells Derived from Naive T Cells Can Utilize a Non-Perforin and Non-FasL Pathway To Inhibit Allogeneic Progenitor Cell Function Ex-Vivo.

Blood ◽

10.1182/blood.v106.11.3029.3029 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3029-3029

Author(s):

Zachary F. Zimmerman ◽

Monica V. Jones ◽

Stephen A. Stohlman ◽

Robert B. Levy

Keyword(s):

T Cells ◽

Progenitor Cell ◽

Ex Vivo ◽

Cell Populations ◽

Naive T Cells ◽

Effector Cells ◽

Naïve T Cells ◽

Effector Pathways ◽

Dependent Pathway

Abstract We are interested in understanding the effector pathways used by host T cells to inhibit engraftment following allogeneic HCT. Previous work in MHC matched models found that mice deficient in either perforin or fasl continue to mediate resistance, however, the numbers of BMCs necessary to override the host barrier is less than in cytotoxically normal mice. These findings suggest both cytotoxic pathways are likely to contribute to the barrier. Notably, in the combined absence of both pathways, resistance is virtually absent. In contrast, animals which contain memory CD8 T cells can resist large numbers of BMCs regardless of the absence of both perforin and fasl pathways. The present studies examined the use of these pathways by T cell populations with respect to the inhibition of progenitor cell (PC)activity (CFU-IL3) from BMC ex-vivo to determine the relative contributions of perforin and/or fasL in inhibition mediated by effector cells derived from naive vs. memory cell populations. Effector cells were generated from unsensitized BALB/c and BALB/c-perf−/− mice as well as mice immunized against H2b alloantigens. Primary effectors from unsensitized mice and memory effectors were generated following 5 and 2–3 day MLC cultures, respectively. Effector cells were co-cultured up to 48 hrs. with BMC and then plated with rmIL-3 to elicit CFU formation. Effector cells from unsensitized BALB/c-wt mice inhibited allogeneic B6-wt CFU (>80%) as well as B6-lpr CFU (>70%) formation. Syngeneic BM was not inhibited. These data demonstrate that the perforin dependent pathway is sufficient for inhibition, consistent with in vivo resistance observed in FasL defective recipients. Effector cells generated from unsensitized BALB/c-perf−/− also effectively inhibited B6-wt CFU (>80%), consistent with a fas mediated pathway. However, in contrast to effectors generated from unsensitized BALB/C-wt mice, effector cells generated from BALB/c-perf−/−did not inhibit CFU from B6-lpr BM, suggesting that: 1) both perforin and fasl effector pathways can be utilized to inhibit CFU formation ex-vivo and, 2) in the absence of both pathways, these primary effector cells could not mediate such inhibition. As anticipated, memory effector cells from BALB/c-wt mice exhibited the same pattern as the "naive" effectors (i.e.>90% inhibition) against both B6-wt and B6-lpr BM. This finding is consistent with inhibition by perforin and/or additional effector pathways. However, memory effector cells from cytotoxically deficient BALB/c-perf−/− mice failed to mediate the same pattern of inhibition as "naive" effectors from this strain. In contrast to the inability of primary effectors to inhibit B6-lpr CFU activity, the memory effectors efficiently (i.e.>90% inhibition) inhibited CFU generation from B6-lpr BM. These findings demonstrate that unlike effector cells generated from naive T cells, memory effectors can use a non-perforin/fasL dependent pathway to inhibit allogeneic PC activity. The results are consistent with the inhibition of engraftment in vivo following conditioning and transplant into cytotoxically double (perforin + fasL) defective mice and support the notion that memory - but not primary effectors utilize a non-perforin/fasl pathway to inhibit allogeneic CFU PC function. Current experiments are examining the kinetics of CFU inhibition and precise characterization of the effector T cells.

Download Full-text