Final Report Summary - STEMS (Pre-clinical evaluation of stem cell therapy in stroke)
Given their expected capacity to self-renew and differentiate efficiently into the desired cell type, clonal populations of Stem cells (SC) promise to produce beneficial effects in a number of diseases. Several studies already indicate that SC transplantation has a therapeutic potential in stroke, with sources of SC that include embryonic, foetal and adult SC, and lines derived from teratoma. To date, two cell types of human origin have been used for SC therapy in stroke, bone marrow-derived SC and cell lines. The NT2N line was derived from a human testicular germ cell tumour. NT2N cells have an exclusive commitment to the neural lineage after retinoic acid treatment, and have been extensively validated in pre-clinical models of stroke without any teratoma or tumour formation reported. A Phase I trial involving 12 patients demonstrated the safety, and partial functional improvements in some patients. A Phase II study showed a global trend towards improvement. The ReN001 cell line, was developed by ReNeuron from human fetal neuroblasts immortalised with the c-mycERTAM technology. A pilot trial with 12 patients is planned in collaboration with the Glasgow University Hospital (United Kingdom).
These studies converge to confirm the feasibility and safety of SC therapy in stroke patients. They also demonstrate the need for developing improvements both at cell source and patients recruitment levels. All cell sources come with advantages and disadvantages that should be evaluated. In fact, much crucial information is still lacking before SC transplantation becomes a clinical reality. For instance, the standardisation of the conditions to regulate SC proliferation and differentiation to produce region-specific grafts need to be better defined; changes in their properties induced by transplantation into lesioned brain structures are poorly understood, as is the full extent of functional improvement at long-term post-stroke delays.
The STEMS project specifically aimed at determining the extent and limits of SC therapy in stroke in order to pave the way for clinical therapeutic trials. This objective had to be achieved through the completion of six successive and complementary tasks:
(1) the definition of the standard experimental conditions for proliferation, guided differentiation, and mass production of SC-derived products for transplantation;
(2) the identification of the best transplantation protocol with regards to location, number of transplanted cells, and post-stroke delays, in a rat model of focal ischemia;
(3) the control of safety and compatibility aspects;
(4) the quantification of the effects of SC transplantation on functional impairments;
(5) the transposition of the optimal experimental conditions defined in rodents to non-human primates;
(6) the definition of the relevant human cell therapy product and operating procedures to be applied to stroke patients.
Work performed and results achieved
This project aimed at defining the potential of SC therapy in stroke and, therefore, had a strong pre-clinical orientation. Accordingly, the work plan was organised as a longitudinal multidisciplinary study implemented from human stem cell culture to the analysis of graft fate and effects in animal models of stroke with non-invasive approaches similar to those used in humans. The main secondary objectives were to:
- define the most efficient cell source, by comparing human embryonic (hESC) and human adult (hANSC) stem cells;
- define the optimal differentiation stage for transplantation;
- define gender differences;
- develop the proof-of-concept of stem cell therapy for stroke at non-human primate level.
The first 18-months could be divided in three periods with clear objectives. The first three months of the work plan have resulted in a major achievement that was not perceived as such initially, the standardisation of procedures among partners. It was quickly apparent that, although bearing the same name (ES cell culture protocol, transient middle cerebral artery occlusion (tMCAO) in rats, animal strains, behavioural tests), the protocols used in our consortium varied so that it was difficult to compare results and reach constructive conclusions. Standardise of protocols was achieved through exchanges of researcher between groups (three one-week stays, one two-weeks stay). This has been beneficial not only for the STEMS project, but also for the international competitiveness of our groups and for European stem cell research as a whole. Exchanges and regular meetings particularly benefited to our young fellows that could start building long-term relationships for their future carrier in neurosciences.
This three-months period was also devoted to establish management guidelines, develop the website, elaborate the graphic chart and publish leaflets, and to obtain the necessary legal authorisations to perform the work.
During the next 15 months, up to the first activity report, two main objectives were reached, the development of a robust protocol for differentiation of human ESC-derived neural progenitors, and the characterisation of a genuine human ANSC line, the NNC1 line provided by partner 2.
During this 18-months period, three events occurred that resulted in modifications of the workplan. Firstly, the NNC1 line turned out to be a pericyte-derived, pluripotent cell line, with low capacity to engage into the neural lineage to form neurons. Secondly, in 2006, Takahashi and Yamanaka had described the possibility to derived pluripotent SC (iPS) from murine somatic (therefore adult) cells. The iPS field developed exponentially during the next two years and we considered that iPS cells can be compared to ANSC. In May 2008, the steering committee decided to include hiPS into the workplan in order to fulfill the comparison with hESC. Thirdly, the primate studies started with an important delay due to moving of CEA to a new site. A six-months prolongation of the STEMS contract was requested to the EU and obtained. In spite of these adjustments, the overall objectives were not modified.
These studies converge to confirm the feasibility and safety of SC therapy in stroke patients. They also demonstrate the need for developing improvements both at cell source and patients recruitment levels. All cell sources come with advantages and disadvantages that should be evaluated. In fact, much crucial information is still lacking before SC transplantation becomes a clinical reality. For instance, the standardisation of the conditions to regulate SC proliferation and differentiation to produce region-specific grafts need to be better defined; changes in their properties induced by transplantation into lesioned brain structures are poorly understood, as is the full extent of functional improvement at long-term post-stroke delays.
The STEMS project specifically aimed at determining the extent and limits of SC therapy in stroke in order to pave the way for clinical therapeutic trials. This objective had to be achieved through the completion of six successive and complementary tasks:
(1) the definition of the standard experimental conditions for proliferation, guided differentiation, and mass production of SC-derived products for transplantation;
(2) the identification of the best transplantation protocol with regards to location, number of transplanted cells, and post-stroke delays, in a rat model of focal ischemia;
(3) the control of safety and compatibility aspects;
(4) the quantification of the effects of SC transplantation on functional impairments;
(5) the transposition of the optimal experimental conditions defined in rodents to non-human primates;
(6) the definition of the relevant human cell therapy product and operating procedures to be applied to stroke patients.
Work performed and results achieved
This project aimed at defining the potential of SC therapy in stroke and, therefore, had a strong pre-clinical orientation. Accordingly, the work plan was organised as a longitudinal multidisciplinary study implemented from human stem cell culture to the analysis of graft fate and effects in animal models of stroke with non-invasive approaches similar to those used in humans. The main secondary objectives were to:
- define the most efficient cell source, by comparing human embryonic (hESC) and human adult (hANSC) stem cells;
- define the optimal differentiation stage for transplantation;
- define gender differences;
- develop the proof-of-concept of stem cell therapy for stroke at non-human primate level.
The first 18-months could be divided in three periods with clear objectives. The first three months of the work plan have resulted in a major achievement that was not perceived as such initially, the standardisation of procedures among partners. It was quickly apparent that, although bearing the same name (ES cell culture protocol, transient middle cerebral artery occlusion (tMCAO) in rats, animal strains, behavioural tests), the protocols used in our consortium varied so that it was difficult to compare results and reach constructive conclusions. Standardise of protocols was achieved through exchanges of researcher between groups (three one-week stays, one two-weeks stay). This has been beneficial not only for the STEMS project, but also for the international competitiveness of our groups and for European stem cell research as a whole. Exchanges and regular meetings particularly benefited to our young fellows that could start building long-term relationships for their future carrier in neurosciences.
This three-months period was also devoted to establish management guidelines, develop the website, elaborate the graphic chart and publish leaflets, and to obtain the necessary legal authorisations to perform the work.
During the next 15 months, up to the first activity report, two main objectives were reached, the development of a robust protocol for differentiation of human ESC-derived neural progenitors, and the characterisation of a genuine human ANSC line, the NNC1 line provided by partner 2.
During this 18-months period, three events occurred that resulted in modifications of the workplan. Firstly, the NNC1 line turned out to be a pericyte-derived, pluripotent cell line, with low capacity to engage into the neural lineage to form neurons. Secondly, in 2006, Takahashi and Yamanaka had described the possibility to derived pluripotent SC (iPS) from murine somatic (therefore adult) cells. The iPS field developed exponentially during the next two years and we considered that iPS cells can be compared to ANSC. In May 2008, the steering committee decided to include hiPS into the workplan in order to fulfill the comparison with hESC. Thirdly, the primate studies started with an important delay due to moving of CEA to a new site. A six-months prolongation of the STEMS contract was requested to the EU and obtained. In spite of these adjustments, the overall objectives were not modified.
