Autologous cell therapy as a new approach to treatment of radiation-induced bone marrow aplasia: preliminary study in a baboon model

The sparing of viable hematopoietic stem and progenitor cells located in underexposed bone marrow territories associated with the relative radioresistance of certain stem cell populations is the rationale for autologous cell therapy consisting of ex vivo expansion of residual cells after collection postirradiation. The feasibility of this treatment mainly depends on time constraints and hematopoietic cell threshold. We showed in this study that in the absence of early-acting mobilizing agent administration, subliminar amounts of CD34+ cells can be collected (1 × 106 CD34+ cells/100 mL bone marrow or for 1 L apheresis) from 6-Gy gamma globally irradiated baboons. Residual CD34+ cells were successfully expanded in serum-free medium in the presence of antiapoptotic cytokine combination (stem cell factor + FLT-3 ligand + thrombopoietin + interleukin 3, 50 ng/mL each, i.e., 4F): KCD34+ = ×2.8 and ×13.7 (n = 2). Moreover, we demonstrated the short-term neutrophil engraftment potential of a low-size mixed expanded graft (1.5 × 106 final CD34+cells/kg) issued from the coculture of unirradiated (20%) and 2.5-Gy in vitro irradiated (80%) CD34+ cells on an allogeneic stromal cell layer in the presence of 4F. Further preclinical research needs to be performed to clearly establish this therapeutic approach that could be optimized by the early administration of antiapoptotic cytokines.Key words: ex vivo expansion, cytokine, cell therapy, bone marrow aplasia, irradiation, animal model.

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Endothelial cell-derived angiopoietin-like protein 2 supports hematopoietic stem cell activities in bone marrow niches

Blood ◽

10.1182/blood.2021011644 ◽

2021 ◽

Author(s):

Zhuo Yu ◽

Wenqian Yang ◽

Xiaoxiao He ◽

Chiqi Chen ◽

Wenrui Li ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Endothelial Cell ◽

Cell Biology ◽

Ex Vivo ◽

Cell Types ◽

Ex Vivo Expansion ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Bone Marrow Niches

Bone marrow niche cells have been reported to fine-tune HSC stemness via direct interaction or secreted components. Nevertheless, how niche cells control HSC activities remains largely unknown. We previously showed that angiopoietin-like protein 2 (ANGPTL2) can support the ex vivo expansion of HSCs by binding to human leukocyte immunoglobulin-like receptor B2 (LILRB2). However, how ANGPTL2 from specific niche cell types regulates HSC activities under physiological conditions is still not clear. Herein, we generated an Angptl2-flox/flox transgenic mouse line and conditionally deleted Angptl2 expression in several niche cells, including Cdh5+ or Tie2+ endothelial cells, Prx1+ mesenchymal stem cells and Pf4+ megakaryocytes, to evaluate its role in the regulation of HSC fate. Interestingly, we demonstrated that only endothelial cell-derived ANGPTL2 and not ANGPTL2 from other niche cell types plays important roles in supporting repopulation capacity, quiescent status and niche localization. Mechanistically, ANGPTL2 enhances PPARD expression to transactivate G0s2 to sustain the perinuclear localization of nucleolin to prevent HSCs from entering the cell cycle. These findings reveal that endothelial cell-derived ANGPTL2 serves as a critical niche component to maintain HSC stemness, which may benefit the understanding of stem cell biology in bone marrow niches and the development of a unique strategy for the ex vivo expansion of HSCs.

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Ex Vivo Expansion of SCID Leukemia-Initiating Cells Using NANEX Nanofiber Scaffold

Blood ◽

10.1182/blood.v118.21.4994.4994 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4994-4994

Author(s):

Stephen L Fischer ◽

Yukang Zhao ◽

Cheng-Kui Qu ◽

Ramasamy Sakthivel

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Drug Development ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Leukemic Cells ◽

Cell Phenotype ◽

Hematopoietic Stem ◽

Starting Point

Abstract Abstract 4994 The concept of the leukemic stem cell (LSC) has gained wide acceptance since it was first definitively established using a NOD-SCID xenotransplantation model nearly 15 years ago. LSCs, which are functionally defined as SCID leukemia-initiating cells (SL-ICs), are believed to possess biological properties that render them resistant to conventional chemotherapy. Although there is still much debate over how to phenotypically define LSCs, there is general agreement that LSCs are rare in acute myeloid leukemia (AML), which has hindered efforts to develop LSC-targeted therapies. In order to provide researchers and pharmaceutical companies with an ample supply of LSCs for testing, methods are needed to generate large numbers of LSCs from patient samples. Ex vivo expansion of LSCs in culture is one approach that offers tremendous promise for increasing cell numbers for research and drug development. Conditions that enable efficient expansion of normal hematopoietic stem cells (HSCs) can be used as a starting point for developing an optimal culture system for LSCs. The natural bone marrow microenvironment maintains HSCs in close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D NANEX nanofiber based cell culture substrate. The NANEX substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSC proliferation. Here, we present our recent work with the NANEX platform towards achieving a high yield ex vivo expansion of LSCs. Using common LSC markers, including CD34, CD38, CD117, and CD123, we quantify and characterize NANEX-expanded leukemic cells using flow cytometry. We compare NANEX to standard tissue culture polystyrene and demonstrate that NANEXÔ significantly improves LSC expansion and reduces clonogenic phenotype loss during ex vivo culture. Additionally, we show that NANEXÔ-expanded cells engraft in NOD-SCID mice and, through limiting dilution analysis, quantify the increase in SL-ICs as a result of culture on NANEXÔ. Our data indicates that NANEX technology provides a robust ex vivo expansion of SL-ICs and, with further development, offers great potential for use in LSC-targeted drug development. Disclosures: No relevant conflicts of interest to declare.

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The Ex-Vivo Expansion of Megakaryocytic Progenitors from Hematopoietic Stem Cells for Thrombocytopenia in NOD/SCID Mice

Blood ◽

10.1182/blood.v128.22.3746.3746 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3746-3746

Author(s):

Mo Yang ◽

Jieyu Ye ◽

Enyu Liang ◽

Chunfu Li ◽

Beng H Chong

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Transplantation ◽

Growth Factor ◽

Cord Blood ◽

Ex Vivo ◽

Cell Types ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Objective: Thrombocytopenia is a common clinical problem in patients with cancer or bone marrow transplantation. Currently it is mainly managed by platelet transfusion. Repeated platelet transfusions are associated with the risks of transfusion reactions/alloimmunisation and may lead to platelet refractoriness. Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is other strategy for the treatment of thrombocytopenia. This study aimed to establish efficient conditions for the expansion of the MK progenitors from enriched CD34(+) cells of umbilical cord blood. Methods: This study investigated the effect of flt-3 ligang (FL), stem cell factor (SCF) and platelet-derived growth factor (PDGF) in combination with other megakaryocyte-promoting cytokines such as thrombopoietin (TPO) on the differentiation and proliferation of megakaryocytic progenitors. As an early acting growth factor, FL may promote the ex vivo expansion of hematopoietic stem and progenitor cells. We compared the effects of FL and SCF in combination with other megakaryocyte-promoting cytokines in megakaryocytic progenitors. Results: In liquid cultures of enriched CD34+ cells from human umbilical cord blood for 14 days, FL plus TPO, interleukin-3 (IL-3), and IL-6 promoted the expansion of nucleated cells, CD34+ cells, CD34+ CD38- cells, and megakaryocyte colony-forming units (CFU-MK) by 300 +/- 115-, 23.8 +/- 11.3-, 33.9 +/- 28.6-, and 584 +/- 220-fold, respectively. Replacing FL with SCF significantly decreased the yield of all cell types. While one human acute lymphoblastic leukemia sample expressed high levels of flt-3 receptor, the four megakaryocytic cell lines (Meg-01, CHRF-288-11, M-07e, and Dami) did not show any positive expression. Our data suggest that the effect of FL in augmenting the expansion of MK progenitors might be due to the early action of FL at the pluripotent stem cell stage. Our results also demonstrated that TPO alone produced a high proportion of CD61(+)CD41(+) cells but a low total cell count and high cell death, resulting in an inferior expansion. The addition of in IL-1 beta, FL and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines produced efficient expansions of CFU-MK up to 400-fold with the highest yield observed in the presence of TPO, IL-1 beta, IL-3, IL-6 and FL. CD34(+) cells were expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61(+)CD41(+) cells, CFU-MK and CD34(+) cells increased by 101%, 134% and 70%, respectively. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in NOD/SCID mice. Conclusions: This study showed that the present cytokine combination and expansion conditions provide an effective and potentially useful system for the clinical expansion of cord blood for bone marrow transplantation (BMT). PDGF might be a suitable growth factor to improve the ex vivo expansion of MK progenitors for clinical applications. Disclosures Yang: National Natural Science Foundation of China: Other: National Natural Science Foundation of China(81270580).

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Characterization of Insulin-Secreting Porcine Bone Marrow Stromal Cells Ex Vivo and Autologous Cell Therapy in Vivo

Cell Transplantation ◽

10.3727/096368914x679363 ◽

2015 ◽

Vol 24 (7) ◽

pp. 1205-1220 ◽

Cited By ~ 4

Author(s):

Hai Van Thi Do ◽

Wan Ting Loke ◽

Irene Kee ◽

Vivienne Liang ◽

Sebastian J. David ◽

...

Keyword(s):

Bone Marrow ◽

Cell Therapy ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Ex Vivo ◽

Marrow Stromal Cells ◽

Autologous Cell ◽

Autologous Cell Therapy

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Recombinant TAT-HOXB4 Protein Promotes Ex Vivo Expansion of Primitive Human Hematopoietic Cells.

Blood ◽

10.1182/blood.v104.11.2855.2855 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2855-2855

Author(s):

Gorazd Krosl ◽

Marie-Pier Giard ◽

Jana Krosl ◽

R. Keith Humphries ◽

Guy Sauvageau ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mononuclear Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Cell Populations ◽

Mouse Bone Marrow ◽

Hematopoietic Stem

Abstract The clinical application of therapeutic protocols depending on hematopoietic stem cell (HSC) transplantation for long term reconstitution with donor-derived HSCs, particularly in patients previously exposed to intensive radiation or chemo-therapy, or when grafts are purged of infiltrating malignant or alloreactive T cells, can be severely hampered by limited numbers of HSCs in the graft. In mouse bone marrow transplantation models, engineered overexpression of HOXB4 has been one of the most potent stimulator of HSC expansion identified to date. The simple addition of soluble recombinant TAT-HOXB4 protein was also recently reported to enable rapid in vitro expansion of mouse HSCs that retain their in vivo proliferation and differentiation capacity. To test the feasibility of using TAT-HOXB4 as a stimulator of human HSC expansion, we performed a series of experiments using CD34+ populations isolated from healthy volunteers. The CD34+ cell populations were cultured in X-Vivo medium supplemented with Stem Cell Factor (300 ng/mL) and G-CSF (50 ng/mL) in the presence or absence of TAT-HOXB4 protein (50 nmol/L) for 4 days. In response to TAT-HOXB4, total numbers of mononuclear cells demonstrated a modest but distinct 2-fold increase compared to controls. TAT-HOXB4 treatment had the largest proliferation enhancing effect on more primitive cell populations such as CFU-GEMM, BFU-E and BFU-Meg, whose numbers increased 26.5 ± 1.4 fold (mean±S.D.), 2.2 ± 0.7 fold and 2.1 ± 0.2 fold, respectively, over their input values, and 19.1 ± 1.3 fold, 2.7 ± 0.7 and 31 ± 3.4 fold, respectively, compared to growth factor only controls. In response to TAT-HOXB4, the total numbers of CD34+CD38-Lin- cells increased 2.1 ± 0.7 fold above their starting numbers compared to a 1.5 ± 0.5 fold loss of this population in control cultures. HSC numbers were enumerated at the beginning, and after a 4-day TAT-HOXB4 treatment period using a NOD/SCID repopulation assay. In response to 50 nM TAT-HOXB4, NOD/SCID repopulating cell (SRC) numbers increased ~2-fold over their input values, compared to a 9-fold loss in control cultures without TAT-HOXB4. These results show that recombinant TAT-HOXB4 protein has the capacity to rapidly induce ex vivo expansion of primitive human bone marrow populations, and suggest that optimization of treatment conditions will rapidly lead to clinically useful expansion of human HSCs.

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Towards the Development of a Closed, Nanofiber-Based Culture System for Clinical Expansion of Cord Blood-Derived CD34+ Cells

Blood ◽

10.1182/blood.v120.21.4411.4411 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4411-4411

Author(s):

Stephen E Fischer ◽

Yiwei Ma ◽

Caitlin Smith ◽

Anirudhasingh Sodha ◽

Yukang Zhao

Keyword(s):

Bone Marrow ◽

Tissue Culture ◽

Stem Cell ◽

Cord Blood ◽

Culture System ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Cell Phenotype ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 4411 Interest in ex vivo hematopoietic stem and progenitor cell (HSPC) expansion has increased in recent years due to the growing importance of these cells in the treatment of a variety of both malignant and non-malignant diseases. Ex vivo expansion of cord blood-derived cells has been particularly investigated because cord is a valuable and readily available source of HSPCs, yet contains limited numbers of cells in each unit. Despite these efforts, most attempts to use expanded cord blood HSPCs in the clinic have been unsuccessful due to the generation of insufficient numbers of cells with the appropriate phenotype and the ability to function in vivo. In many ex vivo culture systems, HSPCs are cultured as a suspension cells and cultured in the presence of various media additives that act to enhance cell proliferation while reducing differentiation. An often-overlooked factor influencing fate decisions is the interaction of HSPCs with a substrate. In the natural bone marrow microenvironment, HSPCs maintain close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D nanofiber-based cell culture substrate (NANEX™). The functionalized NANEX™ substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSPC proliferation while minimizing differentiation. Here, we present our recent work towards developing a closed, NANEX™-based platform for large-scale clinical expansions of cord blood-derived CD34+ cells. We demonstrate that NANEX™ expands CD34+ cells from cord an average of more than 150-fold in 10 day culture, which is at least 2-fold higher than that obtained in standard tissue culture plates. Additionally, we show an approximately 1.5-fold higher proliferation of colony forming cells and a significantly higher engraftment rate in NSG mice for NANEX™-expanded cells compared to cells cultured in tissue culture plates. Furthermore, we demonstrate that the NANEX™ scaffold maintains its HSPC growth promoting characteristics after processing into a closed culture system and offers significant advantages over other culture platforms typically used for HSPC expansions in the clinic (culture bags and T-flasks). Our data indicates that NANEX™ technology provides a robust ex vivo expansion of cord blood HSPCs and, with further development, offers great potential for clinical applications requiring large numbers of functional cells. Disclosures: No relevant conflicts of interest to declare.

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Ex vivo expansion of human hematopoietic stem and progenitor cells

Blood ◽

10.1182/blood-2011-01-283606 ◽

2011 ◽

Vol 117 (23) ◽

pp. 6083-6090 ◽

Cited By ~ 187

Author(s):

Ann Dahlberg ◽

Colleen Delaney ◽

Irwin D. Bernstein

Keyword(s):

Stem Cell ◽

Growth Factors ◽

Notch Signaling ◽

Progenitor Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Growth Factors ◽

Self Renewal ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

AbstractDespite progress in our understanding of the growth factors that support the progressive maturation of the various cell lineages of the hematopoietic system, less is known about factors that govern the self-renewal of hematopoietic stem and progenitor cells (HSPCs), and our ability to expand human HSPC numbers ex vivo remains limited. Interest in stem cell expansion has been heightened by the increasing importance of HSCs in the treatment of both malignant and nonmalignant diseases, as well as their use in gene therapy. To date, most attempts to ex vivo expand HSPCs have used hematopoietic growth factors but have not achieved clinically relevant effects. More recent approaches, including our studies in which activation of the Notch signaling pathway has enabled a clinically relevant ex vivo expansion of HSPCs, have led to renewed interest in this arena. Here we briefly review early attempts at ex vivo expansion by cytokine stimulation followed by an examination of our studies investigating the role of Notch signaling in HSPC self-renewal. We will also review other recently developed approaches for ex vivo expansion, primarily focused on the more extensively studied cord blood–derived stem cell. Finally, we discuss some of the challenges still facing this field.

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