Towards culturing granulocytes: lessons learned from ...

Transfusion medicine: focus on granulocytes

Towards culturing granulocytes: lessons learned from culturing reticulocytes

C.E. Severn1 A.M. Toye1,2 1

School of Biochemistry, Medical Science Building and 2Bristol Institute of Transfusion Sciences, Bristol, UK Correspondence: Ashley Mark Toye E-mail: [email protected]

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There is considerable research interest in the manufacture of blood products from donor stem cells. Red blood cells (RBC) represent a particularly attractive regenerative medicine product because they lack a nucleus and therefore pose minimal risk in terms of potential for transformation in the recipient. However, numbers of RBC required for a normal adult therapeutic dose (2x1012) is still challenging, but encouraging progress has already been made in RBC production from stem cells. White blood cells such as neutrophils represent an alternative blood product that could also be manufactured from stem cells. Granulocytes (a mix of white blood cells) are used to treat patients with neutropenia, and are derived from leukapheresis or by pooling the buffy coats from multiple whole blood donations. Therefore, assuming that neutrophil production can be scaled up to generate an effective dosage, the production of these cells may prove to be an achievable blood product using currently available culture technologies. The short lifespan of neutrophils however, remains an issue that requires attention. This article will make a brief assessment of the current approaches used in the culture of RBC and the feasibility of neutrophil production. Learning goals

At the conclusion of this activity, participants should be able to provide: - an update on the status of the production of reticulocytes from different stem cell sources; - a brief overview of the clinical uses of granulocytes and the potential of manufacturing neutrophils as a blood product; - an update on the issue of scaling up blood production using different techniques, including 3D culture on scaffolds.

The production of therapeutic quantities of cultured red blood cells (cRBCs) is the ultimate goal for numerous blood service and scientific laboratories worldwide, and the success of these endeavours has huge potential for improving healthcare. The attraction is the potential availability of bulk cultured “universal” red blood cells “on tap”, alleviating the deficiency in supply of rare blood groups. This would improve availability of blood for patients such as those with alloimmunization reactions due to multiple transfusions, for treatment of diseases such as sickle cell disease and b-thalassemia. Already, a proof of principle cRBC mini-transfusion has been conducted in a single volunteer illustrating that the cultured RBC survive in circulation and are safe for use in humans.1 The challenge now is to more robustly demonstrate that cRBC are equivalent, with the possibility of prolonged survival due to being reticulocytes, as opposed to circulating RBC of different ages gifted by blood donors. We also need to determine ways to scale up cRBC production to generate clinical quantities of cRBC in a more efficient and cost effective way. Whilst striving to achieve our goal of generating vast quantities of cRBC, it is worth considering that, since all blood components orig-

Introduction

inate from hematopoietic stem cell (HSC) in the bone marrow,2 some of the breakthroughs and technologies developed for manufacturing cRBC will have the potential to be exploited or adapted for production of other blood products. Furthermore, the cellular dose needed for an effective adult dose of granulocytes, for example, is 100-fold lower than cRBC, making this blood cell product attractive.3 This review will briefly summarize recent developments in producing cRBCs with a focus on the potential for culturing granulocytes for clinical use.

In vitro culture of HSCs and other stem cell sources to reticulocytes

Under steady state conditions, the human body efficiently generates approximately 2 million RBC per second in the bone marrow from a tiny contingent of HSCs.4 To do this, the HSC undergoes symmetrical or asymmetrical division to generate daughter cells that eventually expand and differentiate to generate the range of blood cells that populate the blood.5-8 Once committed to the erythroid lineage, these stem cells must transit through a series of expansion and differentiation steps generating the nascent reticulocyte. The reticulocyte then exits the bone marrow and matures in the circulation for two days to pro-

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duce the characteristic biconcave erythrocyte. Over the last 10-15 years, multiple laboratories have developed 2D liquid culture systems that reproduce the process and stages of human erythropoiesis. To date, reticulocytes have been generated with a variable level of efficiency from multiple human stem cell sources, including HSCs from adult peripheral blood or bone marrow,1,9-12 umbilical cord blood13,14 embryonic cells15,16 or induced pluripotent stem cells.17 Several of the culture protocols reported have successfully generated large numbers of reticulocytes.1,9,10,18 These culture systems have been utilized to improve our understanding of erythropoiesis and the assembly of the specialized red blood cell membrane in health and disease.9,19-22 Two key studies have succeeded in manufacturing 2.5 mL and 5 mL of packed leukafiltered cRBC, in the absence of stromal cells. The first utilized stem cells obtained by leukapheresis from a volunteer treated with granulocyte-colony stimulating factor (G-CSF),1 and the second was generated from normal donor cells harvested from the natural wastage from a single donor apheresis cone.9 Both studies conducted a detailed characterization of the cells produced. The cells were confirmed as reticulocytes not erythrocytes, with a larger mean cell volume, lower deformability at high shear stress, the presence of residual cellular organelles, and possession of low levels of surface proteins not normally found in circulating erythrocytes (CD36,CD71,CD98). There were also no unexpected differences in blood group antigen expression observed. Since these cells are morphologically similar to reticulocytes that are normally present in the circulation, the cRBC are anticipated to circulate and function normally in the peripheral circulation and should remodel to erythrocytes, as they were observed to do when injected into a NOD/SCID mouse model.1 Importantly, the reticulocytes were demonstrated to be stable in a modified version of SAGM, a storage medium used for storage of red blood cells, illustrating that the cRBC can be stored for at least four weeks.1 The Paris research group Cr51-labeled the cultured adult human reticulocytes and these were given back to the volunteer from whom they were cultured. The majority of the reticulocytes survived in the circulation after 26 days.1 These data provide evidence of the feasibility of deriving functional human red cells for clinical use through laboratory culture, but a larger trial in more volunteers is needed, followed by a dose escalation study. Red cells generated in culture from other stem cell sources have yet to be fully characterized. Cord blood represents a more abundant source of hematopoietic progenitors than adult blood23 and the existence of national cord stem cell banks means access to material from a potential greater diversity of ethnicities. Reticulocytes can be obtained from cord in larger quantities than from adult blood, but the cells obtained express fetal hemoglobin (HbF) rather than adult globin (HbA). The presence of fetal hemoglobin is not anticipated to be a problem, as there are a variety of hematology patients known to have persistence of high levels of fetal hemoglobin. An alternative is to induce adult levels of β globin using KLF1 and BCL11A-XL.24 A full clinical assessment and comparison of cRBCs produced from cord blood and adults in volunteers is needed to determine whether such manoeuvres are necessary. hESC and hiPS are also attractive as a starting material

for blood cell production due to the high proliferative potential of the cells and are theoretically capable of generating large numbers of red blood cells. Embryonic cells in vivo generate mostly nucleated erythrocytes.25 Depending on the stage at which they are harvested, in vitro cultured embryonic cells can express either embryonic globin genes resembling primitive yolk sac derived erythroblasts or, after extended co-culture with immortilized fetal hepatocytes and can produce developmentally mature fetal-liver like erythroblasts, which express mostly fetal hemoglobin and can enucleate.26 IPS cells derived from fibroblasts or produced from adult or cord peripheral blood mononuclear cells resemble cord blood-derived cells, expressing largely HbF with low levels of β globin.27 Only modest degrees of enucleation and adult hemoglobin expression have been reported thus far using these stem cell sources. Importantly, a comparison of the proteomes of iPSC erythroblasts differentiated from several sources with erythroblasts generated from adult or cord blood have shown these are highly similar with the expected contingent of erythroid-specific membrane proteins. However, differences in cytoskeletal proteins were observed, which may explain the substantially lower enucleation rates achieved with iPSCs.27 An alternative strategy is the use of immortalized erythroid progenitor cell lines obtained by transformation, for example using inducible expression of the human papilloma virus 16 proteins HPV16-E6/E7.28 Since the final red blood cell cellular product lacks a nucleus, the use of such immortalization techniques on adult erythroblasts holds much promise for the future. However, manufacturing of products from transformed cellular starting material would require a careful comparison with normal red blood cells to ensure compatibility and confirm the lack of neoantigens. Any cellular product produced from transformed cells would also need to be irradiated.

Granulocytes for treatment of neutropenia and the production of granulocytes from the 2D culture of stem cells

Neutrophils are very short-lived professional phagocytic cells that are rapidly recruited to sites of tissue damage or microbial invasion where they execute a number of functionalities including engulfment and killing. A deficiency in neutrophils is known as neutropenia, and unfortunately, there is often a period of clinically significant neutropenia after transplantation of hematopoietic stem cells following myeloablative chemotherapy treatment due to the time it takes for restoration of hematopoiesis. Current transfusion approaches to address this use either PBMCs obtained by leukapheresis from donors treated with G-CSF and/or dexamethasone (posing some potential risks for the donor) or a pooled granulocyte product derived from multiple whole blood donations. It is noted that the benefits of these treatments have not been fully established.29 Neutrophils have already been successfully produced ex vivo from a number of different sources including adult or cord CD34+ stem cells,30-34 embryonic stem cells35,36 and iPSCs,37,38 using combinations of cytokines including stem cell factor (SCF), fms-like tyrosine kinase 3 (Flt3) and GCSF. The yield of neutrophils manufactured to date are still substantially lower than the numbers needed for a

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therapeutic dose, so further work is needed. Encouragingly, ex vivo generated neutrophils performed well in the majority of functional tests in terms of respiratory burst, migratory and phagocytic activity. One study observed that ex vivo cultured cells had reduced bactericidal activity, but this could be due to other factors such as culture conditions (if the cells were not fully mature).33 Another hurdle for neutrophil culture is their short life span; this is a particular logistical challenge for neutrophil production, as currently they would need to be harvested and utilized immediately. Hence, an effective means of storage needs to be identified to allow these cells to be kept in stasis, or alternatively, the cultures could be harvested and transfused at a more immature progenitor stage or combinations of longer-lived white blood cells could be cultured, e.g. inclusion of macrophages or monocytes. The majority of data on the subject of scale up currently in the literature is split into two very separate but related goals. The first is the goal of expansion of the CD34+ stem cells to increase the stem cell dose for bone marrow transplantation. The second has the dual goal of expansion and differentiation of blood cells. The latter has largely been achieved using static cultures at laboratory scale or using spinner flasks. Current liquid bioreactor culture methods could theoretically provide enough cells for an estimated minimal granulocyte adult therapeutic dose (2¥1010); however, more work is needed on the bulk expansion potential of stem cells and their efficient differentiation into neutrophils. To manufacture the number of cRBC required for a therapeutic dose of 2¥1012 cells in a conventional erythrocyte transfusion, more efficient culture methods are required; currently erythroid cultures need to be maintained at low density to reduce the chances of spontaneous differentiation, thereby increasing expansion capacity, and the original stem cell input is exhausted. Timmins et al.39 report a log-mean expansion of a 2.25¥108-fold increase in cord blood erythroid cultures grown for 33 days up to a volume of 1 liter. Cells were initially grown at low density in static cultures before transfer to a wave-type bioreactor.39 This level of expansion, if replicated in cultures on a larger scale would facilitate the production of therapeutic quantities of cRBCs. Hollow fiber bioreactors similar to those used for monoclonal antibody production have also been utilized to produce a large numbers of erythroid cells, but the number of reticulocytes were not reported.40 The human body is able to effectively compartmentalize the RBC manufacturing process in the bone marrow, with a tiny contingent of HSCs maintained in a delicate balance of self renewal in the endosteal niche.41 Even a partial replication of the natural process using bioengineered scaffolds has the potential to significantly reduce the cost of RBC manufacture: due to the potential for an increase in RBC production per input of HSC, reduction in labor costs as cells can be harvested via continual egress by using altered adherence during maturation, and importantly, a reduction in media volumes and in the less dependence on expensive cytokines (see Anstee et al. for a more detailed review of scaffolds and erythroid culture41). As yet, there is no clear consensus as to the best material for the scaffold, cell seeding density or medium composi-

Scaling up: bioreactors versus scaffolds

tion, making it difficult to directly compare the different protocols reported. A wide variety of approaches have been used from “lab on a chip” scale with the ability to fine tune media changes to influence individual stem cell expansion, to a variety of static and perfused bioreactors. One approach used is to seed HSCs onto scaffolds in the absence of stromal cells which has achieved substantial expansion of CD34+ cells.40 Another approach is to reconstitute both HSC and stromal cell components of the bone marrow niche by seeding total mononuclear cells from bone marrow or cord blood onto 3D scaffolds. Using this strategy, early progenitors were maintained for between 14-28 days on highly porous polyurethane foam,42,43 PVF resin,44 hydrogels,45 fibrin scaffolds46 or non-woven polyester fiber disks47 to a greater extent than a standard 2D suspension culture. By coupling a perfusion system for cell seeding and culture with a hydroxyapatite scaffold, early progenitors increased over 19 days by more than 100 times compared to 2D culture.48 Long-term maintenance and expansion of CD34+ cells has been demonstrated in a non-woven fabric scaffold for 7-9 weeks49 and in a bioderived bone scaffold for five weeks.50 Microcarriers have also been incorporated into systems to create a 3D environment and have been reported to increase numbers of late erythroblasts.51 Finally, 3D systems have also been utilized for the culture of iPSCs, with Lei et al. demonstrating that hydrogels can successfully expand and allow restriction to several hematopoietic lineages.52

Summary

There is currently a tremendous amount of interest and effort being invested into the manufacture of stem cell derived blood products. The production of RBC represents an attractive cellular product and we are getting ever closer to generating enough cells for an adult therapeutic dose. It is possible that we have set our cell numbers target unnecessarily high by trying to match the cell numbers provided by traditional blood donations, since cRBC represent cells of equivalent age rather than a mixture of ages. Granulocytes are required at far lower therapeutic doses than RBC, which may be achievable using current culturing technology, but further work is needed to investigate expansion of stem cells and neutrophil progenitors and their differentiation into mature neutrophils. Alongside this work, improvements in neutrophil storage are required to extend the product lifespan. 1. Giarratana MC, Rouard H, Dumont A, Kiger L, Safeukui I, Le Pennec PY, et al. Proof of principle for transfusion of in vitro-generated red blood cells. Blood. 2011;118:5071-9. 2. Doulatov S, Notta F, Laurenti E, Dick JE. Hematopoiesis: a human perspective. Cell Stem Cell. 2012;10:120-36. 3. Timmins NE, Nielsen LK. Blood cell manufacture: current methods and future challenges. Trends Biotechnol. 2009;27:415-22. 4. Lemischka IR. Microenvironmental regulation of hematopoietic stem cells. Stem Cells. 1997;15 Suppl 1,63-8. 5. Wang LD, Wagers AJ. Dynamic niches in the origination and differentiation of haematopoietic stem cells. Nat Rev Mol Cell Biol. 2011;12:643-55. 6. Morrison SJ, Scadden DT. The bone marrow niche for haematopoietic stem cells. Nature. 2014;505:327-34. 7. Shen Y, Nilsson SK. Bone, microenvironment and hematopoiesis. Curr Opin Hematol. 2012;19:250-5.

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