Ex Vivo Expansion of CD34+ Cells From Cord Blood, Bone Marrow, and Mobilized Peripheral Blood Using NANEX Nanofiber Scaffold

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4816-4816
Author(s):  
Stephen L Fischer ◽  
Jacqueline M Fonseca ◽  
Yukang Zhao ◽  
Linda L. Kelley ◽  
Ramasamy Sakthivel
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Cell Phenotype ◽  
Cd34 Cells ◽  
Ex Vivo Culture ◽  
Mobilized Peripheral Blood ◽  
Stem Cell Phenotype

Abstract Abstract 4816 Hematopoietic stem cell (HSC) transplantation has become the standard of care for patients with hematologic cancers, anemia, and a variety of other malignant and non-malignant disorders, with greater than 50,000 such procedures being performed globally each year, according to the Worldwide Network for Blood and Marrow Transplantation. Although mobilized peripheral blood (MPB) has become a preferred source of HSCs for transplants, bone marrow (BM) and umbilical cord blood (UCB) are also frequently utilized. Regardless of source, several groups have reported that grafts containing lower total nucleated cell (TNC) and CD34+ cell doses contribute to delayed engraftment and higher graft failure rate. Therefore, methods to increase the total cell number while maintaining the progenitor phenotype, especially the CD34+ progenitor cells, from individual grafts would have a significant clinical impact. Ex vivo expansion of HSCs prior to transplantation is one approach that offers tremendous promise for increasing cell doses and improving clinical outcomes. In many ex vivo culture systems, HSCs 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 HSCs with a substrate. In the natural bone marrow microenvironment, HSCs 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 NANEX nanofiber based cell culture substrate. The functionalized NANEX substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSC proliferation while maintain the progenitors stem cell phenotype. Here, we present our recent work with the NANEX platform towards comparing and achieving a high yield ex vivo expansion of CD34+ cells from MPB, BM, and UCB. Additionally, through the use of flow cytometry and CFU assays, we quantify and characterize NANEX-expanded cells from each source. Furthermore, we compared NANEX to a variety of commercially available products and demonstrate that NANEX significantly improves expansion and reduces phenotype loss during ex vivo culture. Our data indicates that NANEX technology provides a robust ex vivo expansion of HSCs and, with further GMP and clinical development, offers great potential for clinical applications. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

Ex Vivo Expansion of Human Mobilized Peripheral Blood Stem Cells Using Epigenetic Modifiers

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1920-1920
Author(s):  
Santosh Saraf ◽  
Hiroto Araki ◽  
Benjamin Petro ◽  
Kazumi G Yoshinaga ◽  
Simona Taioli ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Transcript Levels ◽  
Epigenetic Modifiers ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood

Abstract Abstract 1920 Currently, a significant percentage of hematopoietic stem cell (HSC) transplantations are being performed using growth factor mobilized peripheral blood (MPB) grafts. Unfortunately, about 5 to 40% of patients are unable to benefit from HSC transplantation due to failure to mobilize and harvest an adequate graft (> 2 × 106 CD34+ cells/kg). Epigenetic modifications are thought to be important in determining the fate of HSC including self renewal and differentiation. We have previously demonstrated that sequential addition of chromatin modifying agents (CMA), 5-aza-2'-deoxyctidine (5azaD) and trichostatin A (TSA), is capable of expanding transplantable HSC 7-fold from human cord blood (CB), likely by preventing the silencing of genes which promote HSC self renewal divisions (Araki et al. Blood 2007). Using the same protocol we have also previously shown that 5azaD/TSA can expand CD34+CD90+ cells containing in vivo repopulating capacity from human bone marrow (BM) 2.5-fold (Milhem et al. Blood 2004). The objectives of our current studies were to assess whether CMA can also expand HSCs present in MPB. In order to test this hypothesis, CD34+ cells were isolated from MPB products from three healthy donors and were expanded ex vivo using 5azaD/TSA for 9 days as described previously (Araki et al. Blood 2007). Following culture, expansion of primitive CD34+CD90+ cells, colony forming unit mixed lineages (CFU-mix), and long term (5 weeks) cobblestone area forming cells (CAFC) were assessed. A 3.74 ± 0.77 fold expansion of CD34+CD90+ cells was observed in 5azaD/TSA expanded MPB cells while only a 0.93 ± 0.23 fold expansion was observed in control cultures (p = 0.025). The 5azaD/TSA expanded MPB cells had a 10.1-fold increase in the number of CFU-mix in comparison to no expansion in the control cultures (p = 0.0055). A 2.26-fold expansion of CAFC numbers was observed in 5azaD/TSA expanded MPB cells in comparison to 0.19-fold expansion in control cultures. Taken together, our data indicate that 5azaD/TSA can expand MPB CD34+CD90+ cells 3.74-fold which also possess the functional capacity to generate primitive CFU-mix and long term CAFCs. This expansion of primitive MPB CD34+CD90+ cells appears to be at an intermediate level (3.74 fold) in comparison to BM and CB which had 2.5-fold and 10.5-fold expansion, respectively. We have previously demonstrated that CD34+CD90+ expanded CB cells are exclusively responsible for reconstituting blood cells following transplantation (Araki et al. Exp Hematol 2006). Currently, the frequency of in vivo repopulating units for CMA expanded MPB is being determined in contrast to expanded BM and CB cells. However, it remains to be investigated what determines the limit for ex vivo expansion of HSC by epigenetic modifiers based on their ontogeny. Towards this goal we analyzed transcription levels of several genes implicated for HSC self renewal/expansion including HoxB4, GATA 2, and Ezh2, which were compared between MPB cells prior to and following expansion in 5azaD/TSA or control cultures. Significantly higher transcript levels were detected for HoxB4 (p = 0.003), GATA 2 (p = 0.0002), and Ezh2 (p = 0.0001) by real time quantitative RT PCR in the 5azaD/TSA expanded MPB graft in comparison to control cultures. Interestingly the transcript levels of HoxB4 and GATA 2 but not Ezh2 were significantly lower in expanded cells in contrast to unmanipulated primary MPB cells. This is in sharp contrast to our earlier results from CB in which 5azaD/TSA expanded cells displayed much higher transcript levels of HoxB4 and GATA 2 compared to primary unmanipulated CB cells. Previously we have demonstrated that environmental conditions can influence the degree of expansion of transplantable HSC from CB (Araki et al. Exp Hematol 2009). Using the same protocol we expanded MPB cells in the presence or absence of CMA using either optimal (SCF, TPO, FLT3L) or suboptimal cytokine cocktails (SCF, TPO, FLT3L with IL-3 and IL-6). Interestingly, unlike CB cells no significant difference in expansion between the two cytokine groups with or without CMA was observed (4.5 versus 4.3-fold expansion of CD34+CD90+ cells, respectively). Corresponding to this, transcript levels of HoxB4 and Ezh2 did not vary between MPB cells expanded with 5azaD/TSA in the two different cytokine environments. Our studies have the potential to be used to expand HSC from poor mobilizers in order to optimize MPB grafts for transplantation. Disclosures: No relevant conflicts of interest to declare.

Viability in Late Stages of Ex Vivo Erythropoiesis Is Enhanced by Increased Cell Density

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4748-4748
Author(s):  
Daniela Boehm ◽  
Mohamed Al-Rubeai ◽  
William G Murphy
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Red Blood Cells ◽  
Cell Density ◽  
Late Stage ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Ex Vivo ◽  
Cd34 Cells ◽  
Late Stages

Abstract Erythropoiesis is one of the body’s most productive cell production processes yielding 2×1011 new red cells from hematopoietic stem cells (HSCs) of the bone marrow every day. Intensive research has focused on mimicking this process ex vivo through application of various growth factor combinations or co-culture with stromal cells. To develop a scalable and reproducible system for large scale production of red blood cells we have investigated in vitro erythropoiesis of peripheral blood derived CD34+ cells with primary focus on the impact of the microenvironment on the process. The influence of cultivation conditions on expansion of erythroid progenitor cells and their terminal differentiation to mature red blood cells were studied in stroma-free liquid culture supplemented with stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO). Peripheral blood derived CD34+ cells were expanded by more than 105 fold over a 3 week period. This degree of expansion has only been achieved previously for CD34+ cells derived from more potent stem cell sources such as cord blood, bone marrow and G-CSF mobilized peripheral blood (Giarratana et al, Nat Biotechnol 2005). The natural environment of human erythropoiesis, the bone marrow, is a very crowded milieu where hematopoietic precursors and other cells are packed in close proximity. Cell crowdedness was found to have significant influences on ex vivo erythropoiesis. Cell density per surface area rather than cell concentration per media volume determined cell expansion during exponential growth where more crowded cells showed reduced overall expansion. In cultures inoculated at 4×105 cells/ml (2.1×105 cells/cm2) increasing cell density per area (i.e. decreasing surface area to volume ratio) 4fold (to 8.4×105 cells/cm2) resulted in 35±12% reduction of total expansion (p<0.05, unpaired Student’s t-test). While 4fold increase of cell density in cultures seeded at 1×106 cells/ml (from 5.3×105 cells/cm2 to 2.1×106 cells/cm2) reduced overall expansion by 51±9% (p<0.01). In late stage erythropoiesis, however, when cells had become arrested in G1 and no longer proliferated, cell density was seen to enhance cell viability. Dilution series of late stage erythroblasts showed that although cell viability gradually decreased over a 14 day cultivation period the decreasing rate was lower in cells cultivated at higher density as shown in the Figure. Enhanced viability in crowded culture conditions could reflect the cells’ dependency on direct cell-cell interactions as found in the marrow environment. Cultures grown to high cell densities of 2–3×106 cells/cm2 showed higher maturation efficiency than previously obtained in this cultivation set-up with more than 80% of cells being CD71-/GpA+. Enucleation yields of up to 45% were achieved indicating a significant amount of terminal maturation to red blood cells. Efficient maturation and particularly enucleation have in many cases been found to be dependent on or improved by interactions with feeder cells or macrophages (Fujimi et al, Int J Hematol 2008). Keeping erythroid cells at high densities during late stages of erythropoiesis possibly helps to mimic their in vivo environment, thus allowing for better survival and efficient terminal maturation without the need for co-culture with other cells. Figure Figure

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

Blood ◽  
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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