Periodic Report Summary 3 - REDONTAP (Proposal for the Continuous Proliferation & Simultaneous Maturation of Haematopoietic Stem Cells into Blood Cell Lineages)
Project Context and Objectives:
Understanding the biological signals and their temporal magnitude involved in the division, maturation and migration of haematopoietic stem cells (HSCs) and their differentiated progeny should allow for a controlled continuous production of mature blood cells. By careful selection of a 3-dimensional micro-environment, it is possible to mimic the niche within bone
marrow in which haematopoiesis occurs. Further, by design and control of the fluid-phase chemical microenvironment and flow profile, it is possible to fine tune the rate of departure of the differentiating cells into a separate microenvironment suitable for further maturation, so creating the conditions for the generation of mature blood cells which could be a continuous process.
This research will determine the requirements for the control of the fluidic and chemical behaviour within different bioreactor compartments for the generation of HSCs. Complex, composite systems that allow for the temporal and spatial separation of microenvironments allowing for not only variation in the fluid flow and oxygen tension, but also changes to the chemical nature of the culture conditions (e.g. access to cytokines and growth factors and essential nutrients) will provide the opportunity for delivery of controlling factors. The principle is based on the ability to provide nutritional exchange with an overall zero, or very small, net mass transport. Mechanical design will allow us to match the rate of HSC division, providing the opportunity to derive the daughter cells into the correct environment for red blood cell development over an appropriate time frame. Differentiation of HSCs into different blood cell types occurs within different bone marrow niches and so mimicry of the erythrocyte niche is likely to result in maximisation of the rate of red blood cell development.
The research aims of the project are:
• To provide a dynamic environment for the asymmetric proliferation of HSCs. Successful attainment of appropriate mode of stem cell proliferation will be determined by enumeration of HSCs, establish that HSC population numbers are retained whilst observing proliferation of haematopoietic daughter cells capable of differentiation into erythrocytes using a standard differentiation protocol;
• To provide continuous perfusion environments for proerythroblast expansion and maturation. Successful attainment of erythrocyte maturation will be determined by the ability of expanded cells to carry oxygen using standard techniques;
• To provide clinically-relevant numbers of functionally mature RBCs which are type-specific. Successful attainment of derivation of type-specific erythrocytes will be determined if it is possible over the maturation life-cycle to produce 1012 functional cells.
The Redontap consortium comprises 4 participants from 4 European countries, one being an SME, one a blood bank and cell and tissue therapy provider in the commercial health care market, supported by two academic institutions:
REDONTAP Project Participants
The University of Liverpool United Kingdom
The University of Leipzig Germany
Applikon Biotechnology BV Netherlands
Banc de Sang i Teixits Spain
Project Results:
(i) Isolation, purification and control of proliferation and differentiation of haematopoietic stem cells
CD34+ mononuclear cells from peripheral blood, umbilical cord blood and bone marrow have been extensively characterised, and baseline phenotypic, genomic, proliferation and erythroid differentiation data produced in various commercial and novel bespoke culture media. By manipulating the cytokine supplementation and altering the cell feeding regimen, we have gained useful improvements in total erythroid CD34+ mononuclear cell numbers.
(ii) Metabolic control of haematopoiesis
We have been successful in identifying previously unknown influences on the efficiency of development of haematopoietic stem/progenitor cells on stroma. High osmolarity has a clear and reproducible effect and is seemingly beneficial for the maintenance of stem/progenitor cells over long periods in stromal culture. At the same time, high osmolarity restricts the outgrowth of the more downstream progenitors responsible for early haematopoietic activity. Our metabolic analyses suggest that an effect of high osmolarity is to bias cellular metabolism towards oxidative mitochondrial respiration and away from glycolysis, so that the combination of reduced glycolytic activity and reduced availability of glucose results in increased dependence on oxygen. This results in the highest retention of long term progenitors, which can be activated into activity by the addition of cytokines after 11 weeks of culture, and so have succeeded in establishing a novel and optimised set of conditions for the support of early progenitors.
(iii) Control of haematopoiesis using cytokines
We were able to achieve high expansion rates of CD34+ umbilical cord blood mononuclear cells that had an elevated progenitor content. There are still technological issues due to large volumes required to obtain clinical doses, but the progenitors were able to differentiate into RBCs, having a high percentage of enucleation. The RBCs obtained seemed to have substantial oxygen uptake capacity.
(iv) Creation of surfaces with tethering peptides
So far, two peptides have been tethered onto gold-coated glass, as a model for specifically-modified bioactive surfaces. Experiments with varying concentrations of these peptides, when attached to the surfaces as part of a self-assembled monolayer of biologically inert peptides, demonstrate a differential response when incubated with whole cord blood and purified CD34+ UCB mononuclear cells. A second set of materials that didn’t rely on condensation of cysteine with gold, namely silane bioconjugation, created lysine-functionalised surfaces that were much more robust. Four separate peptide moieties have been surface-tethered, and all of these have been shown to actively capture cells in flowing media, compared to a static control.
(v) Design and fabrication of bespoke bioreactor
Applikon designed the components required to achieve a working prototype, involving miniaturization of sensors (pH, DO, level, foam, temperature) from 12mm diameter to 6.4mm vessel temperature control systems, flexible OEM controller, and adaptable cultivation modes (batch, fed-batch, continuous and perfusion). This prototype has been rigorously tested in long-term cell culture tests with HSCs at ULIV and BST.
Based on cellular testing at ULIV, which consisted of initial bench testing to determine the usability and reliability of bioreactor features, some aspects of the design were redesigned. These were:
• Uprating of the control of pH, allowing the pH to be raised as well as lowered by adding NaOH in addition to CO2;
• Allowing reduction of dissolved oxygen levels by adding nitrogen to the culture;
• Upgraded software to broaden the control and analysis of parameter-rich culture profiles;
• Reduction in evaporation rate to 3.3% per day, reaching the physical limit possible;
• Scaling-up of the design from 250mL to 500mL to allow continuous perfusion of fresh medium;
Potential Impact:
The proposed research promises to produce a surface capable of capturing and anchoring early haematopoietic progenitor cells, and, by systematically reviewing the possible ways in which to improve efficiency at each stage of the maturation process, provide the best chemical environment for the greatest output of red blood cells. Our process involves the creation of a bespoke multi-chamber bioreactor that retains the early progenitor cells but allows, in a separate chamber, the rapid and extended population expansion of pre-erythroblasts using a different chemical and biomechanical environment. Our research is, therefore, expected to:
• Design and fabricate a surface that can successfully tether early haematopoietic stem cells;
• Design and fabricate a working machine that is capable of converting haematopoietic stem cells into red blood cells;
• Prove the creation of clinical-grade red blood cells using the machine.
1.3.2 Potential impact and use
The objective of the proposal is to allow the on-demand, commercial production of large quantities of erythrocytes without the need to rely on blood donations. In addition, by selection of opportune HSC ABO-H grouping, it will be possible to manufacture specific, including rare, blood groups. Blood transfusion services have the difficulty of providing sufficient quantities of correctly matched units of blood to multiple locations around their nation states. Not only does this present logistical difficulties, but it is very expensive, estimated to cost approximately €3000 per unit of red blood cells. it can be estimated that the annual cost of provision of blood transfusion services within the EC is €30 Billion, and €200 Billion globally. The cost of provision has consistently risen at 30–40% per annum recently, due to patient safety measures. The major risk factors are HIV, hepatitis C and Creutzfeldt-Jakob Disease (CJD) with severe reactions thought to occur in 0.04% of transfusions, purely due to human error of infusion of the incorrect blood type3. Increasing numbers of risk factors continuously limit the supply of donated blood whilst demand generally increases over time. The impact is therefore in a vast reduction in the cost of provision blood supply, and with a concomitant reduction in infection risk to the public.
List of Websites:
http://www.redontap.com
Understanding the biological signals and their temporal magnitude involved in the division, maturation and migration of haematopoietic stem cells (HSCs) and their differentiated progeny should allow for a controlled continuous production of mature blood cells. By careful selection of a 3-dimensional micro-environment, it is possible to mimic the niche within bone
marrow in which haematopoiesis occurs. Further, by design and control of the fluid-phase chemical microenvironment and flow profile, it is possible to fine tune the rate of departure of the differentiating cells into a separate microenvironment suitable for further maturation, so creating the conditions for the generation of mature blood cells which could be a continuous process.
This research will determine the requirements for the control of the fluidic and chemical behaviour within different bioreactor compartments for the generation of HSCs. Complex, composite systems that allow for the temporal and spatial separation of microenvironments allowing for not only variation in the fluid flow and oxygen tension, but also changes to the chemical nature of the culture conditions (e.g. access to cytokines and growth factors and essential nutrients) will provide the opportunity for delivery of controlling factors. The principle is based on the ability to provide nutritional exchange with an overall zero, or very small, net mass transport. Mechanical design will allow us to match the rate of HSC division, providing the opportunity to derive the daughter cells into the correct environment for red blood cell development over an appropriate time frame. Differentiation of HSCs into different blood cell types occurs within different bone marrow niches and so mimicry of the erythrocyte niche is likely to result in maximisation of the rate of red blood cell development.
The research aims of the project are:
• To provide a dynamic environment for the asymmetric proliferation of HSCs. Successful attainment of appropriate mode of stem cell proliferation will be determined by enumeration of HSCs, establish that HSC population numbers are retained whilst observing proliferation of haematopoietic daughter cells capable of differentiation into erythrocytes using a standard differentiation protocol;
• To provide continuous perfusion environments for proerythroblast expansion and maturation. Successful attainment of erythrocyte maturation will be determined by the ability of expanded cells to carry oxygen using standard techniques;
• To provide clinically-relevant numbers of functionally mature RBCs which are type-specific. Successful attainment of derivation of type-specific erythrocytes will be determined if it is possible over the maturation life-cycle to produce 1012 functional cells.
The Redontap consortium comprises 4 participants from 4 European countries, one being an SME, one a blood bank and cell and tissue therapy provider in the commercial health care market, supported by two academic institutions:
REDONTAP Project Participants
The University of Liverpool United Kingdom
The University of Leipzig Germany
Applikon Biotechnology BV Netherlands
Banc de Sang i Teixits Spain
Project Results:
(i) Isolation, purification and control of proliferation and differentiation of haematopoietic stem cells
CD34+ mononuclear cells from peripheral blood, umbilical cord blood and bone marrow have been extensively characterised, and baseline phenotypic, genomic, proliferation and erythroid differentiation data produced in various commercial and novel bespoke culture media. By manipulating the cytokine supplementation and altering the cell feeding regimen, we have gained useful improvements in total erythroid CD34+ mononuclear cell numbers.
(ii) Metabolic control of haematopoiesis
We have been successful in identifying previously unknown influences on the efficiency of development of haematopoietic stem/progenitor cells on stroma. High osmolarity has a clear and reproducible effect and is seemingly beneficial for the maintenance of stem/progenitor cells over long periods in stromal culture. At the same time, high osmolarity restricts the outgrowth of the more downstream progenitors responsible for early haematopoietic activity. Our metabolic analyses suggest that an effect of high osmolarity is to bias cellular metabolism towards oxidative mitochondrial respiration and away from glycolysis, so that the combination of reduced glycolytic activity and reduced availability of glucose results in increased dependence on oxygen. This results in the highest retention of long term progenitors, which can be activated into activity by the addition of cytokines after 11 weeks of culture, and so have succeeded in establishing a novel and optimised set of conditions for the support of early progenitors.
(iii) Control of haematopoiesis using cytokines
We were able to achieve high expansion rates of CD34+ umbilical cord blood mononuclear cells that had an elevated progenitor content. There are still technological issues due to large volumes required to obtain clinical doses, but the progenitors were able to differentiate into RBCs, having a high percentage of enucleation. The RBCs obtained seemed to have substantial oxygen uptake capacity.
(iv) Creation of surfaces with tethering peptides
So far, two peptides have been tethered onto gold-coated glass, as a model for specifically-modified bioactive surfaces. Experiments with varying concentrations of these peptides, when attached to the surfaces as part of a self-assembled monolayer of biologically inert peptides, demonstrate a differential response when incubated with whole cord blood and purified CD34+ UCB mononuclear cells. A second set of materials that didn’t rely on condensation of cysteine with gold, namely silane bioconjugation, created lysine-functionalised surfaces that were much more robust. Four separate peptide moieties have been surface-tethered, and all of these have been shown to actively capture cells in flowing media, compared to a static control.
(v) Design and fabrication of bespoke bioreactor
Applikon designed the components required to achieve a working prototype, involving miniaturization of sensors (pH, DO, level, foam, temperature) from 12mm diameter to 6.4mm vessel temperature control systems, flexible OEM controller, and adaptable cultivation modes (batch, fed-batch, continuous and perfusion). This prototype has been rigorously tested in long-term cell culture tests with HSCs at ULIV and BST.
Based on cellular testing at ULIV, which consisted of initial bench testing to determine the usability and reliability of bioreactor features, some aspects of the design were redesigned. These were:
• Uprating of the control of pH, allowing the pH to be raised as well as lowered by adding NaOH in addition to CO2;
• Allowing reduction of dissolved oxygen levels by adding nitrogen to the culture;
• Upgraded software to broaden the control and analysis of parameter-rich culture profiles;
• Reduction in evaporation rate to 3.3% per day, reaching the physical limit possible;
• Scaling-up of the design from 250mL to 500mL to allow continuous perfusion of fresh medium;
Potential Impact:
The proposed research promises to produce a surface capable of capturing and anchoring early haematopoietic progenitor cells, and, by systematically reviewing the possible ways in which to improve efficiency at each stage of the maturation process, provide the best chemical environment for the greatest output of red blood cells. Our process involves the creation of a bespoke multi-chamber bioreactor that retains the early progenitor cells but allows, in a separate chamber, the rapid and extended population expansion of pre-erythroblasts using a different chemical and biomechanical environment. Our research is, therefore, expected to:
• Design and fabricate a surface that can successfully tether early haematopoietic stem cells;
• Design and fabricate a working machine that is capable of converting haematopoietic stem cells into red blood cells;
• Prove the creation of clinical-grade red blood cells using the machine.
1.3.2 Potential impact and use
The objective of the proposal is to allow the on-demand, commercial production of large quantities of erythrocytes without the need to rely on blood donations. In addition, by selection of opportune HSC ABO-H grouping, it will be possible to manufacture specific, including rare, blood groups. Blood transfusion services have the difficulty of providing sufficient quantities of correctly matched units of blood to multiple locations around their nation states. Not only does this present logistical difficulties, but it is very expensive, estimated to cost approximately €3000 per unit of red blood cells. it can be estimated that the annual cost of provision of blood transfusion services within the EC is €30 Billion, and €200 Billion globally. The cost of provision has consistently risen at 30–40% per annum recently, due to patient safety measures. The major risk factors are HIV, hepatitis C and Creutzfeldt-Jakob Disease (CJD) with severe reactions thought to occur in 0.04% of transfusions, purely due to human error of infusion of the incorrect blood type3. Increasing numbers of risk factors continuously limit the supply of donated blood whilst demand generally increases over time. The impact is therefore in a vast reduction in the cost of provision blood supply, and with a concomitant reduction in infection risk to the public.
List of Websites:
http://www.redontap.com
Other report summaries
- Periodic Report Summary 2 - REDONTAP (Proposal for the Continuous Proliferation & Simultaneous Maturation of Haematopoietic Stem Cells into Blood Cell Lineages)
- Periodic Report Summary - REDONTAP (Proposal for the continuous proliferation & simultaneous maturation of haematopoietic stem cells into blood cell lineages)
- Periodic Report Summary - REDONTAP (Proposal for the continuous proliferation & simultaneous maturation of haematopoietic stem cells into blood cell lineages)