Preliminary Reports of Stereotaxic Stem Cell Transplants in Chronic Stroke Patients

Preclinical studies have suggested that stem cell–based therapies possess the potential to improve stroke outcomes. Recently, the results of two early-stage clinical trials of stem cell therapy for chronic stroke have been published: the United Kingdom–based ReNeuron phase I clinical trial, called “Pilot Investigation of Stem Cells in Stroke” (PISCES, NCT01151124),¹ and the United States–based SanBio phase I/IIa clinical trial, called “A Study of Modified Stem Cells in Stable Ischemic Stroke” (NCT01287936).² ReNeuron employed a genetically engineered neuronal cell line (CTX-DP) derived from human fetal brain, whereas SanBio utilized modified allogeneic mesenchymal stem cells (SB623), derived from bone marrow stromal cells isolated from healthy donors. Both studies reported safety of CTX-DP and SB623 cells at 12–24 months after intracerebral transplantation in chronic stroke patients, but claims of efficacy are limited by the small number of patients. Because CTX-DP and SB623 are genetically modified cells, an in-depth examination of the gene and cell manipulations, and close monitoring of the transplanted patients over the long term are warranted to further assess their safety for cell therapy in stroke.

The CTX-DP immortalized cell line is derived from human first-trimester fetal cortical cells and was genetically modified using c-mycER TAM technology to achieve conditional growth control with a fusion protein comprising a growth-promoting gene, c-myc, and a hormone receptor regulated by the synthetic drug, 4-hydroxytamoxifen.³ SB623 cells exhibit a neuronal phenotype when expanded from human bone marrow mesenchymal stem cells and were transfected with a plasmid expressing the Notch 1 intracellular domain truncated at the transmembrane domain (NICD), which promoted their expansion in the presence of certain trophic factors.⁴ Both CTX-DP and SB623 cells were manufactured as clinical-grade cells for autologous transplantation. The gene manipulation of both cell lines was effectively a mandate from the US Food and Drug Administration (FDA) that highly homogeneous cell populations be used as the graft material for cell transplant therapy for stroke. However, this genetic manipulation nearly brought down both companies, as the trials were delayed nearly 10 years. Both companies initiated their studies in the early 2000s, at a time when gene therapy clinical trials had resulted in several patient deaths.⁵ Because of this, the FDA and the scientific community were cautiously vetting any gene-based therapy, and these two gene-modified stem cell treatments for stroke were subjected to such scrutiny.

The gene modification and cell culture approaches introduced by ReNeuron and SanBio produced highly efficient neuronal cells in vitro.^3,4 Subsequent in vivo studies supported the safety and efficacy of CTX-DP and SB623 following their transplantation into animal models of stroke.^6,7 Moreover, insights into these cells' mechanisms of action have postulated graft-induced endogenous repair processes.^8,9 However, unequivocally silencing or deleting the exogenous inserted genes before transplantation remained a hurdle for UK regulatory and US FDA approvals for CTX-DP and SB623, respectively. To ensure safety of these stem cells, a switch to turn off the gene or a procedure to completely eliminate the gene had to be incorporated into the quality-release protocols for both cell populations. One copy of the conditionally immortalizing c-mycER TAM transgene was integrated into the CTX-DP cell genome under the cytomegalovirus immediate early promoter. CTX-DP cells are clonal, expand rapidly in culture, and exhibit a normal karyotype.³ In cell culture, the c-mycER TAM transgene was silenced by growth arrest (epidermal growth factor, basic fibroblast growth factor, and 4-hydroxytamoxifen withdrawal), prompting the cells to differentiate into neurons and astrocytes.³ CPG methylation serves as the mechanism of silencing following intracerebral implantation of CTX-DP cells into stroke animals.¹⁰

This silencing of the cytomegalovirus transgene promoter in vivo may provide an additional safety feature of transplanted CTX-DP cells. In the case of SB623, human bone marrow mesenchymal stem cells were transfected with a plasmid expressing the human NICD and the neomycin-resistance gene.¹¹ Following gene transfection, G418 selection was applied but stopped after a week, followed by a couple of passages culminating in harvest of the cells using trypsin-EDTA. SB623 cells were routinely characterized by flow cytometry and were found to express mesenchymal features. This transient NICD transfection and selection comprised the safety mechanism.

The approaches of both groups provided an ample supply of well-defined transplantable cells but posed a quandary as to whether the cells retained their optimal therapeutic potential. In particular, some key stemness properties appear to have been sacrificed during the homogenization and neuronal differentiation process; for example, the cells' neuronal fate skewed against the naive cells' capacity to migrate. This meant that the transplant regimen had to cater to the limitations of the final stem cell product rather than the needs of the stroke patient, in that both protocols opted for a surgical maneuver that might exacerbate the injury to the already compromised brain. Partly based on the homogeneity of the CTX-DP and SB623 products, and the historical experience of direct implantation in Parkinson's disease patients,¹² both trials delivered the cells intracerebrally. Although this approach allowed a lower effective dose range of transplantable cells (compared with systemic transplantation) and circumvented the need for the grafted cells to migrate a long distance toward the site of injury, this invasive procedure also necessitated the enrollment of more advanced, chronic stroke patients. Effectively, this resulted in raising the bar for clinical efficacy because of the patients' higher rates of morbidity and mortality.

The ReNeuron PISCES trial was an open-label phase I safety study that enrolled men aged 60 years or older with stable disability based on National Institutes of Health Stroke Scale score ≥6 and modified Rankin Scale score of 2–4.¹ At 6–60 months after ischemic stroke, patients received stereotactic putaminal transplantation of CTX-DP cells. The results showed that single intracerebral doses of CTX-DP cells displayed no cell-related adverse events. Whereas some improvements in neurological and functional outcomes were noted over 24 months post-transplantation,¹ the small patient population of 11 patients with varying stroke onset and the exclusion of women in this trial (to avoid exposure to tamoxifen) limit claims of efficacy in this cohort of transplanted stroke patients.

The SanBio phase I/IIA open-label, single-arm study enrolled 18 patients with stable, chronic stroke.² Safety outcomes included at least one treatment-emergent adverse event in all patients, with six patients exhibiting serious treatment-emergent adverse events probably due to the surgical procedure, but none related to cell treatment and all resolving without further complications. Additionally, there were no reported toxicities or deaths associated with SB623. Functional analyses were limited to 16 patients who completed the 12-month follow-up; thus, although improvements from baseline were detected in European Stroke Scale, Fugl-Meyer total score, and Fugl-Meyer motor function total score, coupled with T₂ fluid-attenuated inversion recovery signal in the ipsilateral cortex 1 week after implantation,² such efficacy readouts warrant closer inspection.

Given that both CTX-DP and SB623 cells are genetically modified cells, it is crucial to evaluate their tumorigenic potential. Close monitoring of the allogeneic transplanted cells via sensitive imaging tools is needed to detect any graft-related adverse events. Direct comparisons of the two trials indicate that safety of the CTX-DP cells was maintained up to 24 months, whereas SB623 was monitored for up to 12 months (follow-up to 24 months is ongoing). The cell doses appear comparable: 2–20 million CTX-DP and 2.5–10 million SB623 cells. The ReNeuron trial was clearly a phase I safety study and the SanBio phase I/IIA was a safety and efficacy study, but as noted above, both enrolled small cohorts of patients, thereby lowering confidence on any claims about efficacy. Both companies have now commenced subsequent trials, with ReNeuron enrolling patients in phase II PISCES II and SanBio proceeding with phase IIb ACTIsSIMA (a study of modified stem cells in patients with chronic motor deficit from ischemic stroke). With the enrollment of additional patients, long-term follow-up, and rigorous assessment of the status of the transplanted cells, the safety and efficacy of stem cell therapy for stroke will be closely evaluated.

These two clinical trials of intracerebral transplants in chronic stroke patients should also be assessed against the backdrop of recent clinical investigations of systemic transplantation of mesenchymal stem cells in acute stroke patients.^13,14 Guidelines for the conduct of preclinical studies and the design of clinical trials are outlined in the Stem Cell Therapeutics as an Emerging Paradigm for Stroke (STEPS) recommendations, with the aim of ensuring both safety and efficacy outcomes.¹⁵ The prevailing stroke pathology largely dictates the cell delivery route of the transplant regimen.¹⁶ The primary ischemic injury acutely upregulates chemoattractants in the brain, allowing minimally invasive intravenous or intra-arterial delivery of stem cells in this early phase of stroke. In contrast, the chronic stroke brain exhibits a tapered chemokine signaling profile that necessitates direct intracerebral implantation of stem cells to the peri-infarct region.¹⁷ Finally, for both direct and peripheral routes of cell delivery, it is paramount to visualize the fate of these transplanted stem cells, not only to detect any untoward tumor or ectopic tissue formation but also to obtain insights into the therapeutic mechanism of action. Although trials such as these are advancing, additional preclinical studies, specifically examining the optimal cell delivery route and the mechanism of action, should allow further optimization of the safety and efficacy of clinical stem cell therapy for stroke.¹⁸

Conflict of Interest

The author receives grant support from SanBio, Inc., Karyopharm, Inc., International Stem Cell Corp., and royalties from Athersys, Inc.