Latest culture techniques: cracking the secrets of bone marrow to mass-produce erythrocytes and platelets ex vivo

Abstract

Since the dawn of medicine, scientists have carefully observed, modeled and interpreted the human body to improve healthcare. At the beginning there were drawings and paintings, now there is three-dimensional modeling. Moving from two-dimensional cultures and towards complex and relevant biomaterials, tissue-engineering approaches have been developed in order to create three-dimensional functional mimics of native organs. The bone marrow represents a challenging organ to reproduce because of its structure and composition that confer it unique biochemical and mechanical features to control hematopoiesis. Reproducing the human bone marrow niche is instrumental to answer the growing demand for human erythrocytes and platelets for fundamental studies and clinical applications in transfusion medicine. In this review, we discuss the latest culture techniques and technological approaches to obtain functional platelets and erythrocytes ex vivo. This is a rapidly evolving field that will define the future of targeted therapies for thrombocytopenia and anemia, but also a long-term promise for new approaches to the understanding and cure of hematologic diseases.

Figure 1.

Bone marrow hematopoiesis. Schematic representation of the adult hematopoietic stem cell niche, showing various cell types and extracellular matrix components that influence the differentiation of blood progenitors. The hierarchical differentiation pathways of megakaryopoiesis and erythropoiesis are highlighted. Megakaryopoiesis is typically characterized by an increase in cell size and ploidy, resulting in the final extension of long pseudopods, called proplatelets, which release platelets into the bloodstream. Erythropoiesis entails several morphological and structural changes that give rise to basophilic, polychromatophilic and acidophilic erythroblasts. At the end of the terminal maturation reticulocytes are released into the bloodstream where they complete their maturation into mature erythrocytes. Mk: megakaryocyte; HSC: hematopoietic stem cell; CMP: common myeloid progenitor; MEP: megakaryocyte-erythroid progenitor. The figure was created using Servier Medical Art templates licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com).

Figure 2.

Overview of sources of stem cells for producing platelets and erythrocytes in vitro. Different stem cell sources have been studied for their potential to generate platelets and erythrocytes in vitro. Primary cells can be obtained either from human or mouse bone marrow, or derived from human peripheral blood and umbilical cord blood hematopoietic stem cells. Furthermore, immortalized cell lines have been generated from human embryonic stem cells, human-induced pluripotent stem cells and adipose-derived mesenchymal stromal/stem cell lines. HSC: hematopoietic stem cells; hESC: human embryonic stem cells; hiPSC: human-induced pluripotent stem cells, ASC: adipose-derived mesenchymal stromal/stem cells. The figure was created using Servier Medical Art templates licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com).

Figure 3.

Overview of soluble factors used in the production of platelets and erythrocytes in vitro. Different cocktails of cytokines, pharmacological agents and/or co-culture with feeder cells have been used to generate platelets and erythrocytes in vitro. HSC: hematopoietic stem cell; FLT3: Fms related tyrosine kinase 3; SCF: stem cell factor; IL: interleukin; IGF-1: insulin-like growth factor-1; TPO: thrombopoietin; EPO: erythropoietin; SR1: stemRegenin 1; AhR: aryl hydrocarbon receptor. The figure was created using Servier Medical Art templates licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com).

Figure 4.

In vivo versus in vitro: an overview of different culture approaches for generating platelets and erythrocytes. In vivo cell maturation occurs in a complex environment in which cells experience different mechanical and biochemical cues due to cell-to-cell and cell-to matrix interactions. In the classical in vitro two-dimensional culture, cell contacts, confinement and environmental biomechanics are lost; moreover cells in contact with the plastic are artificially polarized. In the threedimensional culture, topography and stiffness can be modeled to mimic the native environment. Only cell cultured in flow conditions can recapitulate blood hydrodynamics. Microfluidic devices have the ability to enable extension of proplatelets and the release of functional platelets. Three-dimensional bioreactors combine the advantages of a three-dimensional environment with flow through the scaffold: mature cells can migrate toward the perfused compartment to release either mature erythrocytes or platelets. Cell culture in agitated or stirred-tank bioreactors has been exploited to allow large-scale production of platelets or erythrocytes. 2D: twodimensional; 3D: three-dimensional. The figure was created using Servier Medical Art templates licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com).