Ex Vivo Expanded Megakaryocyte Progenitors Generated From Human Mobilized Peripheral Blood CD34+ Stem Cells Can Be Cryopreserved and Transplanted Into NSG Mice to Generate Functional Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 482-482
Author(s):  
Holger Karsunky ◽  
Robert J. Tressler ◽  
Joy Chananukul
Keyword(s):  
Stem Cells ◽  
Peripheral Blood ◽  
Platelet Transfusion ◽  
Ex Vivo ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Mobilized Peripheral Blood ◽  
Megakaryocyte Progenitors

Abstract Abstract 482 Thrombocytopenia is a common side effect of high-dose chemotherapy that can compromise cancer treatment by requiring treatment delay and/or dose reduction for the patient. Platelet transfusion is typically given to prevent severe hemorrhage. However, several factors including acquisition, banking, and associated risks of bacterial infections and alloimmunization are hampering reliance on platelet transfusion. Growth factors are also used to stimulate proliferation and differentiation of megakaryocytes to increase platelet production, but in severely myelosuppressed patients these have only had modest benefit. The limitations of these two modalities for the treatment of chemotherapy-induced thrombocytopenia indicates that additional treatment approaches are needed. We have developed a novel approach to reconstitute megakaryocytes and platelets in thrombocytopenic patients which is presented here. We have identified a scalable culture system using serum-free medium and a defined cytokine cocktail free of animal products to expand CD34+ hematopoietic stem cells from G-CSF mobilized peripheral blood donors in vitro and direct their development to the megakaryocyte lineage to yield committed human megakaryocyte progenitors (MKPs). These MKPs can be readily cryopreserved while retaining their capacity to generate CFU-MK and platelets in vitro. When infused into NSG mice, ex vivo expanded MKP generate clinically relevant platelet levels of platelets in blood within a few days with sustained platelet levels for several weeks. The platelets generated from MKP in vivo are also functional as assessed by CD62P expression in responses to ADP stimulation in vitro. Our results present a compelling approach for the development of off-the-shelf storable MKPs for the treatment of thrombocytopenia. Disclosures: Karsunky: Cellerant Therapeutics Inc.: Employment, Patents & Royalties. Tressler:Cellerant Therapeutics, Inc.: Employment, Equity Ownership. Chananukul:Cellerant Therapeutics Inc.: Employment, Patents & Royalties.

In Vitro Manipulation of Peripheral Blood Progenitor Cell Collections

1998 ◽  
Vol 21 (6_suppl) ◽  
pp. 1-10
Author(s):  
C. Carlo-Stella ◽  
V. Rizzoli
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Peripheral Blood Progenitor Cells ◽  
Stem And Progenitor Cells ◽  
Therapy Strategies ◽  
Mobilized Peripheral Blood

Mobilized peripheral blood progenitor cells (PBPC) are increasingly used to reconstitute hematopoiesis in patients undergoing high-dose chemoradiotherapy. PBPC collections comprise a heterogeneous population containing both committed progenitors and pluripotent stem cells and can be harvested (i) in steady state, (ii) after chemotherapeutic conditioning, (iii) growth factor priming, or (iv) both. The use of PBPC has opened new therapeutic perspectives mainly related to the availability of large amounts of mobilized hematopoietic stem and progenitor cells. Extensive manipulation of the grafts, including the possibility of exploiting these cells as vehicles for gene therapy strategies, are now possible and will be reviewed.

Granulocyte-Derived Cationic Peptides (GDCPs) Present in Leucophoresis Products Enhance Homing of Hematopoietic Stem Cells (HSCs) to SDF-1 Gradient; Potential Implications for Accelerated Recovery of Hematopoiesis After Transplantation of Mobilized Peripheral Blood Stem Cells (PBSC).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 371-371
Author(s):  
HakMo Lee ◽  
Wan Wu ◽  
Marcin Wysoczynski ◽  
Magdalena Kucia ◽  
Mary J. Laughlin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Cationic Peptides ◽  
Peripheral Blood Stem Cells ◽  
Hematopoietic Stem ◽  
Blood Stem Cells ◽  
Anaphylatoxin C3a ◽  
Mobilized Peripheral Blood

Abstract Abstract 371 Current strategies to accelerate hematopoietic reconstitution after transplantation, include transplantation of greater numbers of HSC or ex vivo expansion of harvested HSC before transplant. However, the number of HSC availabel for allogeneic or autologous transplantation can be low (e.g., umbilical cord blood, poor mobilizers) and strategies to expand HSC and maintain equivalent engraftment capability ex vivo are limited. We reported that some compounds present in leucopheresis products [(e.g., platelet-derived microparticles (Blood 2001, 98: 3143)] and some complement cascade cleavage fragments, e.g., anaphylatoxin C3a (Blood 2005, 101, 3784), enhance the homing responses of HSC to SDF-1 gradient. We recently noted that small cationic peptides released from activated granulocytes (beta2-defensin and cathelicidin) positively prime responsiveness of murine and human HSC to SDF-1 gradient (Leukemia 2009; in press). Accordingly, both compounds enhanced transwell migration of HSC to low threshold doses of SDF-1. This phenomenon was not receptor-dependent, as agonists of membrane receptors that may bind beta2-defensin (FPRL-1), cathelicidin (CCR6) - FPRL-1 agonist, and MIP-3alpha, respectively, did not show similar priming effects. This could be explained by affected distribution of membrane lipids by cationic peptides. In support of this notion, an inhibitor of cell membrane raft formation (methyl-b-cyclodextran) inhibited the priming effect of both compounds, indicating this effect is dependent on CXCR4 incorporation into lipid rafts. Direct confocal analysis of CXCR4 and lipid raft colocalization in the presence or absence of cationic peptides confirmed these findings. Because leucopheresis products are enriched in activated granulocytes that release beta2-defensin and cathelicidin, we tested whether this may explain why mobilized peripheral blood stem cells (PBSC) engraft faster compared to HSC isolated directly from bone marrow (BM) in a murine BM transplant model. Accordingly, syngeneic BMMNCs were exposed ex vivo to beta2-defensin or cathelicidin for 30 minutes and subsequently transplanted into lethally irradiated recipients. We noted that animals transplanted with BM cells primed by those cationic peptides showed accelerated recovery of platelets and neutrophils by ∼3-5 days compared to unprimed control cells. We envision that small cationic peptides, which primarily possess antimicrobial functions and are harmless to mammalian cells, could be clinically applied to prime human HSC before transplantation. This novel approach would be particularly important in cord blood transplantation, where the number of HSC availabel for transplantation is usually limited. We postulate that this promising strategy warrants further investigations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high-dose cyclophosphamide-treated patients: enhancement by intravenous recombinant human granulocyte-macrophage colony-stimulating factor

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 1905-1914 ◽  
Author(s):  
S Siena ◽  
M Bregni ◽  
B Brando ◽  
F Ravagnani ◽  
G Bonadonna ◽  
...  
Keyword(s):  
Stem Cells ◽  
Peripheral Blood ◽  
High Dose ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

We report that hematopoietic progenitor cells expressing the CD34 antigen (CD34+ cells) transiently circulate in the peripheral blood (PB) of cancer patients treated with 7 g/m2 cyclophosphamide (HD-CTX) with or without recombinant human granulocyte macrophage-colony stimulating factor (rHuGM-CSF). In adult humans, CD34+ cells represent a minor fraction (1% to 4%) of bone marrow (BM) cells, comprising virtually all hematopoietic colony-forming progenitors in vitro and probably also stem cells capable of restoring hematopoiesis of lethally irradiated hosts. We show that CD34+ cell circulation is fivefold enhanced by rHuGM-CSF 5.5 protein micrograms/kg/day by continuous intravenous infusion for 14 days after HD-CTX. During the third week after HD-CTX (ie, when CD34+ cells peak in the circulation), large- scale collection of PB leukocytes by three to four continuous-flow leukaphereses allows the yield of 2.19 to 2.73 x 10(9) or 0.45 to 0.56 x 10(9) CD34+ cells depending on whether or not patients receive rHuGM- CSF. The number of CD34+ cells retrieved from the circulation by leukaphereses exceeds the number that can be harvested by multiple BM aspirations under general anesthesia. Thus, after therapy with HD-CTX and rHuGM-CSF, PB represents a rich source of hematopoietic progenitors possibly usable for restoring hematopoiesis after myeloablative chemoradiotherapy. To determine whether CD34+ cells found in the PB are equivalent to their marrow counterpart, we evaluated their in vitro growth characteristics and immunological phenotype by colony assays and dual-color immunofluorescence, respectively. We show that PB CD34+ cells possess qualitatively normal hematopoietic colony growth and high cloning efficiency comparable to that observed with BM CD34+ cells. In addition, PB CD34+ cells display heterogeneous surface membrane differentiation antigens analogous to BM CD34+ cells. The availability of large quantities of CD34+ cells by leukapheresis is relevant to the field of stem cell transplantation and possibly to genetic manipulations of the hematopoietic system in humans.

scholarly journals Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high-dose cyclophosphamide-treated patients: enhancement by intravenous recombinant human granulocyte-macrophage colony-stimulating factor

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 1905-1914 ◽  
Author(s):  
S Siena ◽  
M Bregni ◽  
B Brando ◽  
F Ravagnani ◽  
G Bonadonna ◽  
...  
Keyword(s):  
Stem Cells ◽  
Peripheral Blood ◽  
High Dose ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract We report that hematopoietic progenitor cells expressing the CD34 antigen (CD34+ cells) transiently circulate in the peripheral blood (PB) of cancer patients treated with 7 g/m2 cyclophosphamide (HD-CTX) with or without recombinant human granulocyte macrophage-colony stimulating factor (rHuGM-CSF). In adult humans, CD34+ cells represent a minor fraction (1% to 4%) of bone marrow (BM) cells, comprising virtually all hematopoietic colony-forming progenitors in vitro and probably also stem cells capable of restoring hematopoiesis of lethally irradiated hosts. We show that CD34+ cell circulation is fivefold enhanced by rHuGM-CSF 5.5 protein micrograms/kg/day by continuous intravenous infusion for 14 days after HD-CTX. During the third week after HD-CTX (ie, when CD34+ cells peak in the circulation), large- scale collection of PB leukocytes by three to four continuous-flow leukaphereses allows the yield of 2.19 to 2.73 x 10(9) or 0.45 to 0.56 x 10(9) CD34+ cells depending on whether or not patients receive rHuGM- CSF. The number of CD34+ cells retrieved from the circulation by leukaphereses exceeds the number that can be harvested by multiple BM aspirations under general anesthesia. Thus, after therapy with HD-CTX and rHuGM-CSF, PB represents a rich source of hematopoietic progenitors possibly usable for restoring hematopoiesis after myeloablative chemoradiotherapy. To determine whether CD34+ cells found in the PB are equivalent to their marrow counterpart, we evaluated their in vitro growth characteristics and immunological phenotype by colony assays and dual-color immunofluorescence, respectively. We show that PB CD34+ cells possess qualitatively normal hematopoietic colony growth and high cloning efficiency comparable to that observed with BM CD34+ cells. In addition, PB CD34+ cells display heterogeneous surface membrane differentiation antigens analogous to BM CD34+ cells. The availability of large quantities of CD34+ cells by leukapheresis is relevant to the field of stem cell transplantation and possibly to genetic manipulations of the hematopoietic system in humans.

G-CSF Mobilized Human Mesenchymal Stem Cells Are Found in the Peripheral Blood and Have Telomere Limited Growth Potential.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4246-4246
Author(s):  
Troy C. Lund ◽  
Lisa Baso ◽  
Paul J. Orchard
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Peripheral Blood ◽  
Peripheral Circulation ◽  
Growth Potential ◽  
Hematopoietic Stem ◽  
Marrow Cavity ◽  
Mobilized Peripheral Blood

Abstract Mesenchymal stem cells (MSC) are multipotent cells found lining the bone marrow cavity. Their primary function is to support the growth and differentiation of hematologic progenitors. MSC have been shown to differentiate into a variety of cell types including: bone, adipocytes, cartilage, neuron-like, and muscle-like cells. There is mounting evidence that these cells can, under the right circumstances, enter the peripheral circulation. However, MSC have not been routinely isolated from peripheral blood. Granulocyte colony stimulating factor (G-CSF) is commonly used to mobilize hematopoietic stem cells from the bone marrow into the peripheral circulation. We show that G-CSF mobilized peripheral blood also contains a small percentage of MSC although lower than that of bone marrow derived MSC (BMMSC): 0.012% vs 0.04%. Isolates were morphologically similar to BMMSC and were successfully expanded and shown to differentiate into osteogenic and adipogenic lineages in the appropriate differentiation conditions. FACS analysis showed that the cells reliably expressed cell surfaces markers commonly found on MSC including CD105, CD29, CD166, and CD13. They were negative for CD14, CD34, CD133, and CD45. Mobilized peripheral blood derived MSC (MPB-MSC) had limited expansion potential when compared with bone marrow isolated MSC. Most cells appeared to cease cell division 20–25 days after isolation. MPB-MSC did not have any detectable telomerase activity (as determined by TRAP assay) and consequently were found to have undergone significant telomere shortening (shown by Southern analysis.) The rarity of this cell in G-CSF-mobilized peripheral blood and the subsequent tremendous pressure to divide in cell culture are likely contributing factors leading to the telomere loss seen in MPB-MSC. This phenomenon probably also accounts for the observed senescence observed in vitro. While we have conclusively shown that MSC can be found in G-CSF mobilized peripheral blood, the use of such cells for transplant or gene therapy may be of limited potential due to the telomere-restricted capability of expansion.

scholarly journals Erythropoietic Potential of CD34+ Hematopoietic Stem Cells from Human Cord Blood and G-CSF-Mobilized Peripheral Blood

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Honglian Jin ◽  
Han-Soo Kim ◽  
Sinyoung Kim ◽  
Hyun Ok Kim
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Adult Stem Cells ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood

Red blood cell (RBC) supply for transfusion has been severely constrained by the limited availability of donor blood and the emergence of infection and contamination issues. Alternatively, hematopoietic stem cells (HSCs) from human organs have been increasingly considered as safe and effective blood source. Several methods have been studied to obtain mature RBCs from CD34+ hematopoietic stem cells viain vitroculture. Among them, human cord blood (CB) and granulocyte colony-stimulating factor-mobilized adult peripheral blood (mPB) are common adult stem cells used for allogeneic transplantation. Our present study focuses on comparing CB- and mPB-derived stem cells in differentiation from CD34+ cells into mature RBCs. By using CD34+ cells from cord blood and G-CSF mobilized peripheral blood, we showedin vitroRBC generation of artificial red blood cells. Our results demonstrate that CB- and mPB-derived CD34+ hematopoietic stem cells have similar characteristics when cultured under the same conditions, but differ considerably with respect to expression levels of various genes and hemoglobin development. This study is the first to compare the characteristics of CB- and mPB-derived erythrocytes. The results support the idea that CB and mPB, despite some similarities, possess different erythropoietic potentials inin vitroculture systems.

High-activity samarium-153-EDTMP therapy followed by autologous peripheral blood stem cell support in unresectable osteosarcoma

2001 ◽  
Vol 40 (06) ◽  
pp. 215-220 ◽  
Author(s):  
S. Bielack ◽  
S. Flege ◽  
J. Eckardt ◽  
J. Sciuk ◽  
H. Jürgens ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
High Activity ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cell ◽  
Blood Stem Cell ◽  
High Dose ◽  
External Radiotherapy ◽  
Primary Tumor Site ◽  
Hematopoietic Stem

Summary Purpose: Despite highly efficacious chemotherapy, patients with osteosarcomas still have a poor prognosis if adequate surgical control cannot be obtained. These patients may benefit from therapy with radiolabeled phosphonates. Patients and Methods: Six patients (three male, three female; seven to 41 years) with unresectable primary osteosarcoma (n = 3) or unresectable recurrent sites of osteosarcomas (n = 3) were treated with high-activity of Sm-153-EDTMP (150 MBq/kg BW). In all patients autologous peripheral blood stem cells had been collected before Sm-153-EDTMP therapy. Results: No immediate adverse reactions were observed in the patients. In one patient bone pain increased during the first 48 hrs after therapy. Three patients received pain relief. Autologous peripheral blood stem cell reinfusion was performed on day +12 to +27 in all patients to overcome potentially irreversible damage to the hematopoietic stem cells. In three patient external radiotherapy of the primary tumor site was performed after Sm-153-EDTMP therapy and in two of them polychemotherapy was continued. Thirty-six months later one of these patients is still free of progression. Two further patients are still alive. However, they have developed new metastases. The three patients who had no accompanying external radiotherapy, all died of disease progression five to 20 months after therapy. Conclusion: These preliminary results show that high-dose Sm-153-EDTMP therapy is feasible and warrants further evaluation of efficacy. The combination with external radiation and polychemotherapy seems to be most promising. Although osteosarcoma is believed to be relatively radioresistant, the total focal dose achieved may delay local progression or even achieve permanent local tumor control in patients with surgically inaccessible primary or relapsing tumors.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

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