I've been asked by Dr. Williams who contacted me at my College email to expand our manuscript's explanation of the protocol performed during the course of our clinical trial at Inamdar Hospital in Pune, India. I've provided some key links to our technology, including a link to the patent on the USPTO website (USPTO non-provisional patent 20190117698). The patent contains extensive procedural details in case you'd like to drill down on more information.

Selective Stem Cell Placement

I'll answer your first question regarding selective stem placement and how we developed it in the following way. The research introduces a novel multi-faceted approach known as ‘selective stem cell placement’ (SSCP). This is a proprietary, sequenced regenerative treatment that is based on the hypothesis that in MS, blood brain barrier (BBB) disruption exists in specific relationship to the central vein and the large epiventricular veins. This pathology occurs as a result of distortions and distensions due to a general breakdown of endothelial function. (Schelling, 2014)

Methods for removing and isolating cells from bone marrow are well-known. Through collaboration with specialists and clinicians at Inamdar Hospital in Pune, India, we translated both bench data and methods from other human clinical trials into a novel therapeutic approach beginning with stem cell separation from bone marrow-derived aspirate (BM). The entire protocol was given the name "selective stem cell placement" or SSCP.

After receiving the appropriate accreditation from the local Ethics Committee, we began to recruit participants from a global population in 2011. From a research hospital in Pune, India, we conducted a therapeutic clinical study on patients with multiple sclerosis (MS), over half of whom had either primary or secondary phase MS.

Methodology

As a first step, we draw an amount of BM aspirate from the Iliac Crest (approximately 180-250 mls), the largest source of bone marrow in the body.This BM aspirate sample contains a general population of all stem cell types including a mesenchymal stem cell population (BM-MNSC).

Some additional background on what stem cells do with respect to this methodology is required here. There are many types of stem cells in human bone marrow. Stem cell types found in BM can be characterized morphologically, histochemically and functionally for endothelial/angiogenic cell precursors and neuronal cell precursors. A seminal study published in the 2006 British Journal of Haematology by Dr. Yael Porat entitled “Isolation of adult blood-derived progenitor cell population capable of differentiation into angiogenic, myocardial, and neural lineages” provides evidence that cells differentiate according to their inherent potential. Most every type of cell can differentiate into endothelial cells while only some types of mesenchymal stem cells can differentiate into neurons. Among their many other properties, stem cells have the unique ability to transmigrate toward key stimuli in their local environment in a response known as "chemotaxis." This capacity assures that stem cells get to the points of active inflammation and injury where they can affect regeneration/repair of tissue. Stem cells are also able to communicate with other cell types to marshal them toward areas of injury. Signalling molecules known as "paracrine factors" are emitted to induce responses in receiving cells in order to alter/regulate behaviors.

Medical science is at the beginning of our understanding of the potential of cell-based therapeutic medicine, also known as "regenerative medicine." The potential of regenerative medicine to benefit human health is not yet fully researched or understood. For this study we were mainly concerned with focusing a therapeutic number of stem cells toward known areas of injury within the cranial veins. From there they could differentiate into healthy endothelial cells, which would re-line the inner surfaces of injured veins, thus reestablishing a condition of homeostasis between the central nervous system (CNS) and the BBB — with the blood remaining in the veins where it's supposed to be.

There are other unique aspects to SSCP therapy, including the development of a novel method for selective infusion of stem cells to the areas of continuous and progressive tissue damage deep within the brain. Recent studies have determined that leakage of fibrinogen proteins from injured blood vessels within the central nervous system are the trigger for many similar chronic inflammatory brain disorders. Without a safe and effective way of getting stem cells to the area of injury, this knowledge would have little practical value since the damage is located deep in the cerebral veins.

Fig. 1 Sagittal View of brain lesion alignment with major cerebral veins

That is why only neuro-inflammation due to the MS disease process has been the target of the drug companies throughout the 30-year developmental history of disease modifying drugs (DMD) and biologic immunosuppressive drugs. There are no claims that either drug therapy treats the source of the disease, or good evidence that the timelines of the disease itself change. The manufacturers' only claim that their drugs modify the symptoms of attacks so that they are better-tolerated by the patient.

Your second question of how this clinical trial is different in its approach to "injecting stem cells into the arm" is the key to understanding the difference between our methodology and all other types of MS therapies, whether drugs or the new protocols using stem cells. It also sheds some light on the reason why 30 years of drug solutions have not been particularly effective in treating MS.

I think that what we've uniquely done with respect to the methodology makes a significant difference to the outcome. The treatment comprises a novel sequenced method that induces temporary reverse venous flow in the internal jugular veins IJVs by expanding a balloon and creating an occlusion. Stem cells are then infused by way of a multi-lumen catheter into the reversed flow at the distal end of the IJV, proximal to the sigmoid sinus. (described in detail in the patent). This allows the newly-introduced population of stem cells to reach injured vascular tissue in the large collecting veins (central vein and the epiventricular veins). The therapeutic dose of stem cells, site-specifically delivered to the inner lumen of the cranial veins is capable of repairing and regenerating the damaged, leaking tissue. The proximity of the entire dosage of cells to the area of injury is why we call it "selective placement." The hypothesis is based on the simple premise that you must directly treat a chronically bleeding open wound; you wouldn't put a band-aid 2 inches from a bleeding cut.

By contrast, most clinical methods using stem cells to treat neurologic diseases have adopted a peripheral approach (injection into the patient's arm) using cells derived from the patient's belly fat. Not only do the limited number of stem cells become diluted by systemically transfusing every part of the body, but the population of larger pluripotent stem cells become sequestered in the lungs and cannot get to the area of injury. In fact, not enough cells of any type reach the targeted tissue. Not surprisingly, the results of these therapies, often being performed by plastic surgeons, have not been particularly positive, but they can be done in a few hours and the patients can be on their way. The difference is that in following the evidence, our protocol is complex by necessity, requiring the patient to stay in the hospital for 2 weeks. There are no shortcuts — and perhaps the inherent complexity and high costs are among the reasons why this method has not been proposed before. Whatever the reason, there are no other methodologies world-wide that describe the sequence of procedures necessary for the infused cells to reach deep inside the brain as we have laid out in our protocol.

It might be useful to visualize the conditions in the deep veins that lead to neuroinflammation, subsequent lesions and eventual progressive disability in MS. The image below is taken from Davalos in his description of Experimental Autoimmune Encephalomyelitis, or EAE. The tight, smooth-walled vessel on the left side of the image is how all healthy veins start out. The conditions that lead to MS can be seen progressively moving from left to right. On the extreme right, damage due to trauma has occurred and the blood begins to infiltrate the CNS, triggering the autoimmune response. Pharmaceutical companies developing drugs to treat the inflammation like to advance other theories as to why inflammation occurs. They have promoted everything but existing pathology as the reason for the immune response and subsequent inflammation (leading to de-myelination of axons). In attempting to solve any problem, it's never useful to ignore the evidence. And it's very clear that drug company-sponsored clinical trials have been looking at the wrong things going on in the MS brain for decades.

Fig. 2 Stages in experimental autoimmune encephalomyelitis (EAE)

Knowing that insidious chronic bleeds throughout the central vein are precisely what is happening in MS begs some important basic questions. These are questions that all clinical investigators need to answer if we are to come to consensus on an effective treatment: For example, knowing what we know now, how important would it be to get enough healing stem cells to the open wounds in order to repair the leakage of blood-borne neurotoxins into the brain? — and how do we get them to that point?

Your third question regarding the measurement of morphology and classification of the re-infused cell population is also a good one. We were concerned mainly with infusing enough of every type of cell to an area of endothelial injury. Most every stem cell type can potentially reconstitute vasculature (endothelium) for brain vessel diseases. Only specific cells such as pluripotent and multipotent BM-MNSCs may differentiate into cells required for repairing the damage to neurons in chronic inflammatory demyelinating diseases.

The aim of acute treatment of venous endothelial tissue is to reduce recurrent bleeding. We hypothesized that once we controlled the extravasation, there would be a natural recovery of neuronal tissue, potentially aided by the infusion of a population of BM-MNSCs to the CNS side of the BBB. Many investigators have proven the similarities between post-injury neuroplasticity and the formative events that occur during normal brain development. (Nudo, 2014)

To date, BM stem cell population types are less well understood than researchers admit. For example, how many cells in any measured sample of marrow-derived aspirate are capable of differentiation into neuronal cells? How does gender, age, weight, and health of the patient affect this population? At least one well-known clinical study hypothesis states that all BM-MNCs have the pluripotency to differentiate into neuronal tissue. But this is not the case. And the other answers we don't know either. One of our objectives, therefore, will be to take precise measurements of blood-derived progenitor cell population types in Phase II clinical trials.

Efficacy

No other clinical trials for MS therapies have demonstrated that patients can recover ability as measured by standardized quantitative mobility and leg function performance tests. Since the Phase I trial began in August of 2011, 29 of 31 subjects demonstrated significant functional improvement and recovery of mobility within 10 days.

Additionally, no adverse effects were noted, which echoes the safety findings from previous studies using autologous bone marrow stem cell (BM-MNSC) transplants (Chahine et al., 2016a; 2016b; Sharma et al., 2012; Shyu et al., 2006; Trounson, 2009; Yamout et al., 2010), including transplants through CSF (Oraee-Yazdani et al., 2015).

Our published study, THE EFFECTIVENESS OF SELECTIVE STEM CELL PLACEMENT ON GAIT PERFORMANCE IN PATIENTS WITH MULTIPLE SCLEROSIS: A PHASE I CLINICAL TRIAL, provides evidence for SSCP’s safety and tolerability as a treatment, and rationale for utilizing the same procedure with a larger sample. We note that many study subjects have continued to improve beyond the testing parameters, absent of previous disease symptoms, a subject of future publications.

These outcomes indicate an understanding of disease origins and the respective pathophysiology of the injury necessary to be treated. Because the protocol uses no drugs that produce late-effect risks (such as chemo drugs) and the therapies are minimally invasive and well-tolerated, SSCP may potentially be considered as a first-line treatment.

"We can't go on like this. We have to heal this wound or we will never stop bleeding." - Atticus Finch, in To Kill a Mockingbird

Those words were written by Harper Lee and spoken about the malignant racial inequality in Alabama of the 1930s. But they work even better when they are applied in a literal sense to multiple sclerosis in 2019. Like the white majority desperate to maintain the status quo in southern society, so too the drug industry is determined to protect their turf by ensuring that their sponsored research never looks beyond the inflammatory response in developing new treatments for MS.

We believe that our novel protocol has been poorly-understood because it uses different therapeutic principles than typical drug treatments. The purpose of selective placement, or what otherwise might be known as "drug targeting" is to deliver a pharmacologically effective concentration of the therapeutic agent to the disease site. This treatment, using genetically identical autologous stem cells (the donor being the recipient) that adapt to the local environment where they can specialize, if they were drugs, would be an enhanced definition of the clinical practice of "precision or personalized medicine."

In all respects, this novel protocol is the Occam's razor choice. The philosophical principle of Occam's razor states that one should not make more assumptions than the minimum needed. In other words, the the simplest solution should be the first one to test.

So this is what we know about the clinical course of MS: In the seminal event, we know the veins are bleeding out into the brain. We know that specific blood proteins cause the autoimmune response, inflammation and demyelination of axons, which in turn leads to multiple areas of scarring (sclerosis) in normal looking white matter. We know that areas of scarring correspond precisely to the points where the blood is infiltrating the CNS. We know this sclerosis results in vision loss, muscle weakness, bladder and bowel dysfunction, and progressive disability in patients. We know that stem cells heal damaged endothelial tissue. We know that if we apply the stem cells to the veins to heal injured tissue, there is an immediate response that improves ability and reduces the physical manifestations of the disease. Further study will inform us whether we have reduced the risk of the condition that's causing the recurrent/chronic bleeding and the anomalous immune response. As it is, we believe that SSCP therapy accounts for each link in a chain of events that occurs in the clinical course of MS. Drug solutions account only for the immune response but not the antecedent events that give rise to an increase in recurrent or chronic hemorrhaging. As an important first step we must stop the bleeding.

I hope that answers your 3 questions. Please feel free to email me at hbroeska0@saddleback.edu if you have any further comments or follow-up questions regarding the explanation.