Why produce red blood cells in culture?

Complement existing blood collection systems to support transfusion needs

Red blood cell transfusions, which were developed over 80 years ago, are widely used in emergency medicine and are a crucial component of major surgical procedures, chemotherapy, and the treatment of hereditary anemias, including β-thalassemia major and sickle cell disease (SCD). In developed countries, 1 unit of blood is used per 20 people per year on average, and in the US alone, over 20 million units of RBCs were collected last year.

The number of SCD patients managed with chronic RBC transfusions is increasing as prophylactic transfusions prevent stroke in some children with SCD. A large population of older individuals with myelo displastic syndrome is also chronically transfused and difficult to serve with current collection methods.

In the Western world, the volunteer-based blood collection system usually meets transfusion needs, except for rare blood groups which are often in short supply. In many parts of the developing world, RBC shortages are common due to an insufficiently developed blood collection system. Even in countries with an overall surplus of cells, shortages do occur due to local conditions or a high number of difficult-to-transfuse individuals.

Chronically transfused patients are the most underserved population, as they require precise matching once they have developed allo-antibodies against normally non-reactive blood group antigens. This can lead to shortages, especially for patients with SCD, due to antigen mismatch between the mostly Caucasian RBC donor population and the African-American sickle cell patient population, and the large antigen diversity in African-Americans.

The existing transfusion system is expensive to maintain and vulnerable to major disruptions, such as the emergence of novel pathogens or social upheaval. While short-term blood needs during emergencies can usually be fulfilled by locally available supplies, long-term supply chain disruptions could occur due to a major pandemic, for example. The supply problems and shortages are expected to worsen in the next 20-30 years due to the aging of the Western population and a decrease in the proportion of younger donors.

To address these issues, the in vitro differentiation of RBCs from stem and progenitor cells has been developed as a potential alternative to the current procurement system. The production of cultured RBCs (cRBCs) from stem cells holds the promise of revolutionizing transfusion medicine and the existing RBC supply system. Several groups of researchers are exploring various strategies to produce cRBCs, which would reduce shortages and provide a critical backup capability in case of major emergencies.

Red blood cells as drug delivery vehicles

Blood transfusions have been a commonly used medical treatment for over 80 years. However, there has been growing interest in using red blood cells (RBCs) as more than just carriers of oxygen. This is because drug delivery through RBCs has the potential to increase the half-life of therapeutic agents, limit toxicity and protect drugs from the immune system.

Modifying RBCs: Early efforts to modify RBCs involved changing their surface antigens. to make them more universal and physically loading them with drugs or surface molecules . Despite technical difficulties, these efforts have been successful and multiple clinical trials are underway, demonstrating the potential of this technology. RBCs collected from volunteers have been loaded with therapeutic agents such as asparaginase and decorated with antibodies targeted to glycophorin A.

With advancements in cell culture and stem cell biology, in vitro production of RBCs from genetically modified stem cells has become an alternative strategy for loading RBCs with drugs. This method has the advantage of being able to produce genetically homogeneous cells from a single rare donor, eliminating the risk of contamination and decreasing production complications.

In vitro production of RBCs

RBCs

Primary cells: Adult primary hematopoietic stem and progenitor cells (HSPCs) were the first human cells differentiated into cRBCs in liquid culture. Since then, large progress has been made and it is now possible to expand the HSPCs from one unit of cord or peripheral blood into several units of enucleated RBCs. This could be useful to treat a few patients or to generate small batches of therapeutic RBCs. However, since HSPCs expansion does not eliminate the need to constantly collect cells from volunteers, we and others have developed methods to produce cRBCs from immortal cells such as self-renewing progenitors, immortalized progenitors and iPSCs.

Immortalized cells: Immortalized animal erythroid progenitors were produced over 30 years ago, but the cell lines produced exhibited abnormal karyotypes and enucleated poorly upon terminal differentiation. In addition, the immortalization protocols were not robustly reproducible because they relied on the acquisition of unknown spontaneous mutations in addition to the over-expression of specific oncogenes. More recently, more robust immortalization protocols have been developed that yield lines that can terminally differentiate and enucleate at higher but still relatively modest rates. These lines are an exciting avenue of research and may become an important source of cultured RBCs. However, all lines produced so far are karyotypically unstable, exhibit low growth rate and can only be cultured at low density. Therefore, more progress is necessary before these lines can be used for the production of cRBCs on a large scale.

iPSCs: Human embryonic stem cells were first successfully differentiated into blood cells in 2001. Many researchers, including our team, have improved upon the initial protocol, leading to the ability to produce 100,000 enucleated RBCs per iPSC.

The main advantages of iPSCs are that they are easy to produce and a genetically-modifiable and inexhaustible source of cells for industrial production.

However, iPSCs also have two drawbacks. The first is that they yield cells with an embryonic/fetal phenotype, while immortalized progenitors may yield cells expressing mostly adult hemoglobin. This is a minor drawback for applications where the cRBCs are used as drug carriers, as these cells do not need optimal oxygen delivery capacity to fulfill their function. A more significant drawback of iPSCs is that longer, more complex differentiation protocols are required to produce cRBCs (about 40 days versus 10-20 days for hematopoietic stem and progenitor cells). We have reduced the complexity of the iPSC differentiation method and developed a chemically-defined scalable protocol.

On balance, there are multiple exciting sources of cells to produce cRBCs, and all should be further developed. Our focus is on iPSCs due to our specific expertise, but we also follow the progress of immortalized cells as many of the steps to produce cRBCs are common to both cell sources and can be adapted.

Scaling up

RBCs

A milliliter of blood contains 5 billion red blood cells. Producing RBCs for transfusion support or as therapeutics requires the production of trillions of cells, which is technically challenging as the number of cells needed is at least 2 orders of magnitude larger than for other cell therapies.

In addition to cost, increasing cell concentration in culture is critical to producing high numbers of cells. In the body, RBCs are produced in the bone marrow at concentrations exceeding 1 billion/mL, allowing the production of more than 200 billion RBCs per day in a small volume.

In static culture, the maximum density that can be reached is about 2-3 million/mL. Our team is exploring the use of holofiber reactors to increase cell concentration in our culture to greater than 500 million/mL. The picture illustrate our current system that can be used to produce RBCs equivalent to a few milliliter of blood.