I'm Gary Steinberg at Stanford University, and I'll be discussing stem cell therapy for stroke today. This was originally scheduled as a breakfast seminar for the in-person 2021 AANS annual meeting scientific program, but because of the ongoing COVID pandemic, we're presenting it as a distance learning e-module. I hope that we'll be able to meet in person next year. We first started studying this in the laboratory 20 years ago. We transplanted human fetal neural cells into the rat cortex one week after a stroke and looked at the effect of the stroke brain environment on the cells as well as the effect of the cells on the brain. And you can see here that when we transplanted the cells around the stroke and the cells are stained brown, they migrate to the stroke in a targeted fashion, which is quite interesting because if you place the cells into a brain without a stroke or other injury, the cells don't move. So why do the cells migrate to the stroke area? It's because the cells express chemokine receptors such as CXCR4 that interact with chemokines that are given off by the stroke environment like SDF1 and there are other similar interactions. So the cells that we're injecting are quite clever and we're depending on that to induce the recovery. And you can see here that the cells recover behavior in a rodent model. These are some other cells, also neural stem cells, that recover behavior on several different tests. How do the cells recover neurologic function? The initial notion was that these cells injected into the brain turn into neural stem cells. They then differentiate into the cell types in the brain, including neurons, which would reconstitute neural circuits. That is not how the cells work. We and other groups showed that the cells secrete very powerful trophic factors, growth factors, angiogenesis factors, molecules, and proteins that enhance native mechanisms of recovery known as plasticity. These include endogenous axonal dendritic sprouting, angiogenesis. They have a very powerful effect in terms of immunomodulation and they enhance native neurogenesis, gliogenesis, and synaptogenesis. So in a simple sense, what the cells do is turn the adult brain into a neonatal infant brain that recovers extremely well after stroke or any other kind of injury. Here's a study we did to see whether blocking one of the secreted factors from the human stem cells, VEGF, would affect the recovery. We used Avastin, which blocks human VEGF but not rat VEGF. After stroking the animals, we treated them with the cells alone or the cells plus Avastin. When you deliver Avastin, we do not see the normal recovery when you treat with the cells or the cells plus a nonspecific antibody IgG. We see excellent recovery and we found out that this secreted factor VEGF is important not just for the behavior recovery but also for the induced neovascularization and immunomodulation. This is a rapidly moving field and so far 31 clinical studies have been published of stem cell transplant for ischemic stroke. The vast majority of these studies have used either hematopoietic lineage, bone marrow-derived peripheral blood or umbilical cord blood cells, or mesenchymal stromal lineage from bone marrow umbilical cord or adipose tissue. From bone marrow umbilical cord or adipose tissue, mesenchymal cells, these have used either autologous or allogeneic transplants. There have been a few studies of neural lineage cells used. I've been involved in a number of these studies. All of these studies but two were phase 1 or 1-2a. There was one phase 2b study, one phase 3 study, 12 included controls, 13 were randomized and the temporal window of treatment was between 1 day and 20 years. All of these studies except one were shown to be safe and feasible and some of the studies showed neurologic benefit. The routes of administration of these published studies have included intravenous using between 80 million and 1.2 billion cells injected intravenously. Interarterial using between 10 and 500 million cells and intracranial with 300,000 to 55 million cells. There was one intrathecal study using 60 to 70 million cells. The intravenous route is obviously the easiest and safest but does not get good distribution to the brain. It's not clear whether that matters or not. Interarterial studies have used infusion into the internal carotid or middle cerebral artery and again there's low distribution into the brain but probably better than for intravenous. Intracranial implantation results in the best deposit of cells within the brain. It's more invasive but so far at least in the small studies that have been done it's showing the most impressive results in terms of recovery. Here's a trial that was published in 2011. This was an intravenous autologous bone marrow derived cell study for acute stroke. Patients were treated within 24 to 72 hours after the stroke. 7 to 10 million cells per kilogram were delivered intravenously and there were no significant adverse effects related to the cells. At six months all the patients had improved at least one point on the modified Rankin scale but it's hard to know whether that was due to the cells or the natural recovery because patients recover over the first six months after a stroke. The conclusion was that the cells were safe and feasible in this study. This particular study used an intra-arterial route of delivery again bone marrow mononuclear cells. The study was from Spain in 10 patients and included 10 control patients and you can see there was a trend towards the number of these CD34 positive cells injected and improvement in the Barthel index although it didn't reach statistical significance. Here's a more recent study which was disappointing. This was a sham controlled randomized phase 2 study run by Aldegene and was recently published in Circulation. 29 patients received the intra-arterial injection of the cells versus 19 controls and the results were negative for any benefit. This was an interesting study from Korea. These patients came in with an acute MCA stroke. They were randomized into either receiving cells or the control group. If they were to receive cells they had a bone marrow aspirate and the cells were placed in culture. They were expanded and then 100 million of these cells were injected intravenously between one and two months following the stroke. This is what the strokes looked like. They were large MCA strokes and you can see that the mortality was reduced in the patients who received the cells and furthermore the patients who received the cells showed improvement MRS 0 to 3 to a higher extent than the patients in the control group. This study, the master study which was sponsored by Athercys, was a randomized double-blind placebo-controlled phase 2 study. It was highly anticipated in terms of the outcome results and it utilized a proprietary adult adherent cell population that the company developed. Originally, the patients were to receive cells or placebo intravenously between 24 and 36 hours after the stroke. However, because of slow enrollment the time window was extended to 48 hours and the primary efficacy endpoint was an interesting one. The global test statistic which test statistic which included the modified Rankin score 0 to 2 NIH stroke scale score with a change greater than or equal to 75 percent and the Barthel index greater than equal to 95. For all patients in the study there was no significant difference in the primary outcome or key secondary outcome points so this was considered a negative study. However, if you analyze patients who achieve what's called an excellent outcome this was a post-hoc analysis which was defined as MRS less than or equal to 1 and NIH score less than or equal to 1 with a Barthel index greater than equal to 95. Then there was a benefit for all subjects you could see at 365 days one year statistically significant and then if you included only the patients who were treated within 36 hours a very marked benefit statistically significant. If you look at the odds ratio for both efficacy endpoints here and morbidity and mortality over here you can see that the patients who received the cells within the original time point 24 to 36 hours showed a significant benefit with less morbidity and mortality. This has encouraged the company to plan a phase 3 trial. We worked with a local biotech company SanBio located in Mountain View and did the first intracerebral stem cell stroke trial in North America. This was done at two sites Stanford and University of Pittsburgh and this used adult bone marrow derived stromal stem cells that were harvested from two donors. The cells were placed in culture transiently transfected with a notch gene expanded in culture cryopreserved and then shipped to these sites. What does the transient notch transfection do? It changes the methylation state or differentiation state we don't know if it's necessary but it was used in animal models and therefore carried over to the human stem cell. The notch gene is lost during propagation and passaging of the cells so by the time they're injected into the patients it's unlikely that the plasmid is present. How do the cells work? They do not turn into neural progenitors or the difficult types of cells in the brain. They do not even turn into endothelial cells. What they do is as I described before they secrete very powerful trophic factors, growth factors, antigenesis factors, molecules and proteins that enhance native mechanisms of recovery and perhaps the most important is modulating the immune system to enhance plasticity. They secrete many factors as do other cells including VEGF and other growth factors as well as other cytokines. And the cells are present interestingly in the brains of animals at one month post-transplantation but by two months they're gone so they do not have to stay around for a long time. This is not cell replacement therapy as I've mentioned. Here's how we did the study. This was an open label safety study. We performed it at Stanford and University of Pittsburgh. It was a dose escalation study. We did not use immunosuppression. The reasoning was that the cells themselves are immunosuppressive so the FDA surprisingly allowed that. We placed a stereotactic frame on the patients and then using a burr hole the size of a nickel utilized three needle passage. We placed the cells around the stroke. We didn't transplant directly into the subcortical stroke because it's a very inhospitable environment and with each needle track we utilized five cell implant sites. The patients were 33 to 75 years old. They were seven to 36 months out from their stroke and almost all the patients were years out from their stroke so you knew they weren't going to recover. They've been stable. They've been through rehab. The primary endpoints in this study were safety at two years post implantation but we also had a primary efficacy endpoint which was the European stroke scale at six months post implant and there were a number of secondary outcomes that were evaluated as well. Here's what the strokes looked like. All the patients had to have a subcortical stroke like this but some of the patients had cortical strokes. We targeted the subcortical stroke. The reason that the cortical stroke was not targeted was for fear of inducing seizures and here's how we did the study. All the patients were treated awake under local anesthetic. They were all discharged the day after surgery. It was a dose escalation from two and a half million cells up to 10 million cells. I treated 12 of the patients at Stanford. Six were treated at the University of Pittsburgh. We had very few what are called serious adverse events meaning the patients required a subsequent hospitalization. One patient had a seizure 70 days after the transplant. Not clear it was even due to the surgery. There was one asymptomatic subdural hygroma hematoma I drained even though it was asymptomatic. A pneumonia, a stenting of a cervical carotid artery, unrelated to these cells or to the surgery for cells, a UTI and sepsis, a TIA in a delayed fashion, and in the second year one patient had parasitic dysphagia. None of the patients withdrew to the secondary to these adverse events all resolved without sequela. None were related to the cells only the subdural definitely related to surgery and no correlation between these events in the cell dosage. And here's what the clinical outcomes looked like over two years. We were stunned to see these results. You can observe compared to their baseline patients started improving on the primary outcome ESS score over the first month continued improving at three months. This was statistically significant at the primary endpoint six months and sustained at 12 months as well as at 24 months. This was also true on the other outcome scores. Three quarters of the patients achieved what's called a clinically meaningful recovery, and that signifies that they had a 10 point or more improvement on the Fugl-Miter motor score. This means that the patients could do things they couldn't do before, such as walking, turning doorknobs, or other activities. No relationship between the cell dose and the clinical outcome in this study, and there was no association between the clinical outcome measures at either the baseline stroke severity or the patient's age. And here's one of the patients that we treated. She's one of our older patients, 71 years old. She's two years out after a right basal ganglia stroke from a mitral valve repair at the time we transplanted her. She's paralyzed on the left side. One, two, three. Okay, fine. This is our neurologist examining her. Can you extend it at all? All she can move is her thumb. Yeah, you can. Can't get her leg off the bed. Two, three, four, five. Let go. And as I say, she's been wheelchair bound, been through physical therapy and rehabilitation. After we transplanted her, I went by to see her late that night before going home and asked her to lift her paralyzed arm off the bed and she lifted it over her head. And I wondered if I'd gotten the baseline exam wrong, so I called Neil Schwartz, our neurologist at Stanford who examined her, and said, Neil, you're not gonna believe this. And he said, you're right, I don't believe you. So we decided to go by the next morning and see what she was doing to examine her. And here she is the next morning. Time for it, as hard as you can. To your nose. Oh my goodness. Video it for that. And touch my hand. One, two, three, four, five. That's great, okay. So quite a remarkable result. And I received an email from our neurologist who has given me permission to show this. And the title is, I believe, I must admit it's a bit of a miracle when a neurologist tells you this, you know that something's going on because our neurologists, of course, are appropriately skeptical about recovery, whereas as neurosurgeons, we tend to be more optimistic. And here she is six months later. That's really good. Great. Okay, smile. That's really good. Here she is walking. So we wondered what the mechanism of recovery was and we started doing DTI tractography in our patients. Here's an example of a patient before surgery and after the transplantation and no change in the tractography. But here's something we noticed. This is the MR scan one day post-operatively at a higher level than the stroke in this patient. Looks the same. One week later, there's a new flare lesion in the premotor cortex. It's DWI negative, so it's not a stroke. And then that resolves at two months, never comes back, yet the patient continues to show improved outcome. And lift it up. Here's a second patient. She's 39 years old. She's two years out from her left middle cerebral artery stroke. She was unable to move her right arm. Her speech was not understandable and she had significant problems walking. She did not want to get married because she felt she'd be embarrassed walking down the aisle. Here she is two years after her stroke. And lift it up. Transplant. So she can't move her arm. And here she is two and a half months after transplant. Yay, I'm gonna get you. Ah, I'm gonna get you. She actually gave me an award four and a half years after her transplant at the Smithsonian Conjugability Celebration. For two years after that, I was unable to say more than 20 words. I couldn't move my right arm more than a few inches and could only walk for five minutes without needing a wheelchair. It's been four and a half years to the day now and I'm able to climb stairs, have conversations with family and friends. I run, I work out. My life is amazing. I was also able to become a mother. For two years after that, I was unable to say more than 20 words. I couldn't move my right arm more than a few inches and could only walk for five minutes without needing a wheelchair. It's been four and a half years to the day now and I'm able to climb stairs, have conversations with family and friends. I run, I work out. My life is amazing. I was also able to become a mother. So if this therapy works, even a subpopulation of patients, it will radically improve the outcome for the 7 million patients living in the U.S. alone with disability from chronic stroke. And here's her scan. You can see before the transplant, her stroke is located in the deep subcortical area as well as the cortex. Here's a higher level pre-transplant. And one day after transplant at this higher level, there's a little blood in this sulcus, but nothing significant. One week later, again, that same flare, positive lesion, DWI negative in the premotor cortex area that resolves by two months, never comes back. We had 14 of the 18 patients with this new T2 flare, transient lesion in or adjacent to the premotor cortex. And despite the fact that it was only transient and resolves by one to two months, the size of that initial transient flare lesion post-transplant was highly correlated with the extent of neurologic recovery at 12 months and 24 months post-transplant. So something very important is going on in that region. What we learned from this study was that intracerebral transplantation of these human stem cells in chronic stroke patients was both safe and feasible. We saw significant neurologic improvement at six, 12, and 24 months following transplant. But most importantly, we learned that our prior thoughts that these circuits were dead or irreversibly damaged after a stroke are not true and that they can be resurrected. We're still trying to figure out what the mechanisms are. My hypothesis was that the flare lesion represented a beneficial inflammatory response that somehow jump-started the circuits. And I recently received an NIH grant to study this in the laboratory. SanBio then went on to conduct a multicenter phase two double-blind randomized study in traumatic brain injury patients. This was a multicenter study with a six-month follow-up. And this was a positive study. It was recently published in Neurology. Also, SanBio conducted a multicenter chronic stroke multicenter phase two double-blind randomized study of 163 patients. We participated in both these studies, the TBI and the chronic stroke study. The stroke study was disappointingly negative for the primary outcome. However, in a post-hoc analysis of 77 patients with smaller infarcts, it was shown to be positive with 49% of the treatment group improving greater than or equal to nine points on this neurologic outcome scale compared to their baseline. Whereas the control group only achieved that benefit in 19%, and this was statistically significant. I also wanted to mention the Pisces studies that have been done. These used a cell developed by a company in Scotland called Renuron. This is an allogeneic, fetal-derived, conditionally immortalized neural stem cell. 11 patients were treated in the first study between six and 24 months after their stroke. And this was a positive study. In the second study, between six and 24 months after their stroke, they had stable unilateral hemiparesis and the outcome was compared to their baseline. You can see there was improvement in many of the patients and this was encouraging enough that it led to a second study. This phase two study was published in 2020 and was conducted in the United Kingdom. The primary endpoint was not met. However, there was significant improvement in the ARET subtest too. And this was encouraging enough that Renuron started a randomized blinded controlled phase three trial in the US. In 2019, we participated in this study as well. The primary outcome was greater than or equal to one point improvement on the MRS at six months. However, in June of 2020, enrollment was suspended and the trial was terminated because of the effect of COVID and a change in direction the company was taking. There are many unresolved issues. We don't know the best cell source. Should the cells come from the brain, from adipose tissue, from bone marrow, from umbilical cord? How many cells? What types of cells? Should they be neural stem cells, mesenchymal cells? Should we immunosuppress? We still don't know the best time to treat. Initially, we thought it would be best to treat in the subacute period because that's where much of the recovery occurs in the animal models that have been studied. However, now that we're seeing some promising results in patients who have chronic stroke, we're targeting that population since there are many more patients who might benefit and patients are not in that phase where they're recovering spontaneously. We don't know the best route of delivery, intravenous, intraarterial, intracerebral, or intrathecal. All of the most dramatic results have been seen with the intracerebral direct transplant into the brain. We're still working on mechanisms of recovery. There are currently 27 clinical trials using transplanted stem cells for ischemic stroke that are either ongoing or not yet published. The vast majority, again, of these are using hematopoietic or mesenchymal lineage. There are a couple using neural lineage. 26 of these are early phase studies. One's a phase three. And the temporal window for treatment is between 18 hours and five years. I don't have time to discuss these trials now. However, I will tell you that we developed a cell line in my laboratory 20 years ago, NR1. These are human embryonic-derived neural stem cells. We found that they recover function in several different independent laboratories. They differentiate mostly into neuronal-type lineage. They do not form tumors. And in the last 13 years, I've spent more than $36 million in grants and philanthropy trying to move these stem cells from my lab into the clinic. And I'm pleased to say that just over a year ago, we received approval from the FDA to conduct a phase one 2A trial with these cells. Two weeks ago, I was awarded a $12 million grant to perform this first in human trial, which will be done at Stanford. It's the only trial currently in North America that will be using intracerebral transplantation of stem cells for chronic stroke. We plan to treat our first patient later this month in September, 2021. However, we want to be cautious and don't want to oversell this type of therapy. There's a lot of hope, but considerable hype regarding stem cell therapy for stroke and other indications. I was part of an international society for stem cell research task force that looked at this, and we want to discourage patients from going offshore and receiving cells. We don't even know what type of cells they are in an unregulated fashion. Here's an example of what can happen. This was a patient with ataxia telangiectasia who received cells injected intracerebellar and intrathecally and four years later developed a glio-neuronal neoplasm. They were transplanted in Moscow. Here's another patient who actually received their own olfactory mucosal cells. They had a spinal cord injury. The cells were transplanted into the spinal cord, and you can see eight years later, they developed a mass. This patient was transplanted in Portugal, and it's not just offshore. Here's what happened in a clinic in our own country. This happened in Florida. This was published three years ago in the New England Journal of Medicine, and three patients who had macular degeneration and were losing their vision but could still see had their own autologous stem cells that were derived from their fat tissue, isolated and injected into their vitreous, and they all went blind. So in conclusion, I believe cell transplantation therapy for stroke holds enormous promise. However, we are still in early stages of investigation, and there are many fundamental issues that still need to be resolved. The initial clinical studies appear feasible and safe with the suggestion of efficacy. Further phase one, two, and three clinical studies should be pursued, but very, very important to include controls. I want to thank the entire cerebrovascular team and Stroke Center at Stanford for the successes we've achieved together over the last 31 years. I want to thank my laboratory members who have done all the work and contributed greatly over the last 20 years, as well as my collaborators in science at Stanford and elsewhere. I hope all of you will stay safe and healthy during this COVID crisis and that it will end soon so we can get back to a semblance of normalcy. Thank you for your attention.

stem cell therapy

stroke

Gary Steinberg

Stanford University

neural cells

clinical trials

recovery mechanisms