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Proof of principle for transfusion of in vitro generated red blood cells

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From Blood
By Stuart P. Atkinson

The chronic lack of donated blood, with an annual requirement of nearly 90 million units worldwide presents a major problem which, with an increasing world population, will only get worse. Apart from typical blood donation, another possible source of blood cells is through in vitro manipulations of stem cell populations, such as circulating haematopoietic stem cells (HSCs) or, potentially, embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs); the latter being particularly attractive for patients with blood-related disease. The advantages of stem cell-derived blood are many; these could potentially provide an unlimited source of the various blood types, and reduce the risk of infectious disease from donated blood. However, to date, the transfusion potential of stem cell-derived blood products generated in vitro has yet to be assessed in man. Now in a study (Giarratana et al) from the group of Luc Douay at UPMC University Paris, France, published online in Blood, these issues have been addressed. They report that in vitro-derived reticulocytes (cultured red blood cells; cRBCs) have a similar functional capacity to native reticulocytes, can mature appropriately in mouse and, importantly, they report the successful transfusion and in vivo survival of cRBCs in a human patient.

The group had previously described a protocol for the ex vivo culture of CD34+ HSCs (Giarratana et al), but as the use of stromal cells did not allow for the scaling up of this process (Timmins and Nielsen), another protocol was sought after. The protocol described for cRBC production without the use of stromal cells consisted of the collection of CD34+ HSCs obtained by leukapheresis after mobilization with G-CSF from peripheral blood, followed by exposure to SCF, IL3 and EPO to ensure cell proliferation and erythroid differentiation. Cells from 9 different donors gave a mean cell amplification of around 61,500 fold for CD34+ cells, and giving 81% of morphologically enucleated cells by day 18 of treatment. These cells displayed reticulocyte characteristics (positive staining for new blue methylene and thiazole orange), with a mean cell volume slightly increased relative to control reticulocytes, due to a phenomena known as induced stress erythropoiesis (Migliaccio et al). They were also immunophenotypically and enzymatically similar to native reticulocytes as demarcated by the appropriate levels of the erythrocyte membrane markers GPA, CD71 (transferring receptor) and CD36 (thrombospondin receptor), G6PD and pyruvate kinase enzymatic activities, and also similar with regards to deformability, essential for the repeated ability to pass through microvessels. Analysis of these cells demonstrated high levels of haemoglobin A (88 ± 2.7%), but also some fetal haemoglobin (10.6 ± 2.8%) which was again suggested to be linked to stress erythropoiesis observed in culture, but which is also promoted by SCF stimulation (Bhanu et al). Sufficiently high levels of fetal haemoglobin could have an impact on cell function by reducing oxygen content and deformability. Functional analysis of cRBC-generated haemoglobin, as measured by tonometry, however found cRBCs to reversibly bind O2 in the same manner as native RBCs and that oxygen affinity was also similar. When the purified cRBCs were stored at +4°C for up to 4 weeks in a Saline Adenine Glucose Mannitol (Sag-M) preservative-based solution, they reacted in a similar manner to stored native reticulocytes. The haemoglobin content of cRBCs was maintained and after 4 weeks of storage while CD71 expression, reticulocyte content and deformability remained close to that of fresh cRBCs.

cRBCs labelled with carboxyfluorescein succinimidyl ester (CFSE) were then transplanted by intraperitoneal injection into immunodeficient NOD/SCID mice, to allow analysis of the in vivo maturation of these cells from reticulocytes into functional discocytes. CFSE+ cells were detected for 5 days post-injection with a peak between days 2 and 3 representing 1.5-20% of the total peripheral blood cells of the animal. CD71 levels significantly decreased, with only 5% of CFSE+ cells being CD71+ and LDS (Laser Dye Styryl, a nucleic acid dye) levels dropped to zero over the first 5 days, altogether suggesting the maturation of the cRBCs. Further, cells sorted on day 3 had the form of a biconcave disk, a feature indispensible for efficient gaseous exchange, suggesting overall that the maturation of cRBCs mirrors that of native reticulocytes, which are known to mature fully between 48 and 72 hours. Additionally, cRBC stored in Sag-M at +4°C for 15 days behaved in a manner comparable to fresh cells in vivo in the NOD/SCID mouse.

This infusion study was then tested in vivo in a single human patient. One million CD34+ cells were expanded to around 4 x 1010 cells with a 68% enucleation rate, and were labelled with 51Cr (which binds to haemoglobin without cell destruction). The survival rate of these cells on the 26th day following infusion ranged from 41% to 63% which compares favourably to the reported half-life of approximately 28 days for native RBCs, demonstrating for the first time the persistence of cRBCs for several weeks in vivo in a human patient and thus establishing the feasibility of the concept of the transfusion of cRBCs.

Overall, this exciting piece of work brings us a step closer towards finding an unlimited source of functional blood. However, questions still remain and further obstacles need to be traversed. One unit of conventional packed blood contains 2 x 1012 RBCs, so the challenge still remains to boost the production level. The authors suggest that cord blood derived HSCs may be the optimal source, as these generate 5 – 10 fold more RBCs than HSCs from peripheral blood, as used within this study. However, the availability of iPSCs or blood type-matched ESCs could also make these suitable candidates. The transplantation study described herein obviously needs to be repeated on a much larger scale, and this report mentions only survival of the cells, but with no functional studies or indicators of effects of the transfused cells to the patient’s wellbeing.

 

References

Bhanu NV, Trice TA, Lee YT, Miller JL.
A signaling mechanism for growth-related expression of fetal haemoglobin.
Blood. 2004 Mar 1;103(5):1929-33..

Giarratana MC, Kobari L, Lapillonne H, Chalmers D, Kiger L, Cynober T, Marden MC, Wajcman H, Douay L.
Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells.
Nat Biotechnol. 2005 Jan;23(1):69-74.

Giarratana MC, Rouard H, Dumont A, Kiger L, Safeukui I, Le Pennec PY, François S, Trugnan G, Peyrard T, Marie T, Jolly S, Hebert N, Mazurier C, Mario N, Harmand L, Lapillonne H, Devaux JY, Douay L.
Proof of principle for transfusion of in vitro generated red blood cells.
Blood. 2011 Sep 1.

Migliaccio AR, Whitsett C, Migliaccio G.
Erythroid cells in vitro: from developmental biology to blood transfusion products.
Curr Opin Hematol. 2009 Jul;16(4):259-68.

Timmins NE, Nielsen LK.
Blood cell manufacture: current methods and future challenges.
Trends Biotechnol. 2009 Jul;27(7):415-22.