Closing gaps, opening doors: an experimental collaboration in stem cell intervention.

Despite years of warnings by the academic community that for most of the stem cell-based therapies offered in the private arena little evidence of efficacy exists, these services have been increasingly offered by Canadian private clinics. Recently, as the culmination of years of clashes between stem cell researchers and therapy providers, Health Canada issued a statement prohibiting any type of cell therapy that is not specifically approved. In this climate of conflict, a small group representing both these communities as well as the government gathered in Vancouver to identify common values, and agree on principles to move forward constructively. This historic moment demonstrated that even in this contentious space a meeting-of-minds in between researchers, clinicians, ethicists, entrepreneurs and other stakeholders is possible.

Delivery of oncolytic vaccinia virus by matched allogeneic stem cells overcomes critical innate and adaptive immune barriers.

BACKGROUND: Previous studies have identified IFNγ as an important early barrier to oncolytic viruses including vaccinia. The existing innate and adaptive immune barriers restricting oncolytic virotherapy, however, can be overcome using autologous or allogeneic mesenchymal stem cells as carrier cells with unique immunosuppressive properties. METHODS: To test the ability of mesenchymal stem cells to overcome innate and adaptive immune barriers and to successfully deliver oncolytic vaccinia virus to tumor cells, we performed flow cytometry and virus plaque assay analysis of ex vivo co-cultures of stem cells infected with vaccinia virus in the presence of peripheral blood mononuclear cells from healthy donors. Comparative analysis was performed to establish statistically significant correlations and to evaluate the effect of stem cells on the activity of key immune cell populations. RESULTS: Here, we demonstrate that adipose-derived stem cells (ADSCs) have the potential to eradicate resistant tumor cells through a combination of potent virus amplification and sensitization of the tumor cells to virus infection. Moreover, the ADSCs demonstrate ability to function as a virus-amplifying Trojan horse in the presence of both autologous and allogeneic human PBMCs, which can be linked to the intrinsic immunosuppressive properties of stem cells and their unique potential to overcome innate and adaptive immune barriers. The clinical application of ready-to-use ex vivo expanded allogeneic stem cell lines, however, appears significantly restricted by patient-specific allogeneic differences associated with the induction of potent anti-stem cell cytotoxic and IFNγ responses. These allogeneic responses originate from both innate (NK)- and adaptive (T)- immune cells and might compromise therapeutic efficacy through direct elimination of the stem cells or the induction of an anti-viral state, which can block the potential of the Trojan horse to amplify and deliver vaccinia virus to the tumor. CONCLUSIONS: Overall, our findings and data indicate the feasibility to establish simple and informative assays that capture critically important patient-specific differences in the immune responses to the virus and stem cells, which allows for proper patient-stem cell matching and enables the effective use of off-the-shelf allogeneic cell-based delivery platforms, thus providing a more practical and commercially viable alternative to the autologous stem cell approach.

First-in-human study of TK-positive oncolytic vaccinia virus delivered by adipose stromal vascular fraction cells.

BACKGROUND: ACAM2000, a thymidine kinase (TK)-positive strain of vaccinia virus, is the current smallpox vaccine in the US. Preclinical testing demonstrated potent oncolytic activity of ACAM2000 against several tumor types. This Phase I clinical trial of ACAM2000 delivered by autologous adipose stromal vascular fraction (SVF) cells was conducted to determine the safety and feasibility of such a treatment in patients with advanced solid tumors or acute myeloid leukemia (AML). METHODS: Twenty-four patients with solid tumors and two patients with AML participated in this open-label, non-randomized dose-escalation trial. All patients were treated with SVF derived from autologous fat and incubated for 15 min to 1 h with ACAM2000 before application. Six patients received systemic intravenous application only, one patient received intra-tumoral application only, 15 patients received combination intravenous with intra-tumoral deployment, 3 patients received intravenous and intra-peritoneal injection and 1 patient received intravenous, intra-tumoral and intra-peritoneal injections. Safety at each dose level of ACAM2000 (1.4 × 106 plaque-forming units (PFU) to 1.8 × 107 PFU) was evaluated. Blood samples for PK assessments, flow cytometry and cytokine analysis were collected at baseline and 1 min, 1 h, 1 day, 1 week, 1 month, 3 months and 6 months following treatment. RESULTS: No serious toxicities (> grade 2) were reported. Seven patients reported an adverse event (AE) in this study: self-limiting skin rashes, lasting 7 to 18 days-an expected adverse reaction to ACAM2000. No AEs leading to study discontinuation were reported. Viral DNA was detected in all patients' blood samples immediately following treatment. Interestingly, in 8 patients viral DNA disappeared 1 day and re-appeared 1 week post treatment, suggesting active viral replication at tumor sites, and correlating with longer survival of these patients. No major increase in cytokine levels or correlation between cytokine levels and skin rashes was noted. We were able to assess some initial efficacy signals, especially when the ACAM2000/SVF treatment was combined with checkpoint inhibition. CONCLUSIONS: Treatment with ACAM2000/SVF in patients with advanced solid tumors or AML is safe and well tolerated, and several patients had signals of an anticancer effect. These promising initial clinical results merit further investigation of therapeutic utility. Trial registration Retrospectively registered (ISRCTN#10201650) on October 22, 2018.

Human intracerebroventricular (ICV) injection of autologous, non-engineered, adipose-derived stromal vascular fraction (ADSVF) for neurodegenerative disorders: results of a 3-year phase 1 study of 113 injections in 31 patients.

We have chosen to test the safety of human intracerebroventricular (ICV) brain injections of autologous non-genetically-modified adipose-derived stromal vascular fraction (ADSVF). In this IRB-approved trial, 24 patients received ICV ADSVF via an implanted reservoir between 5/22/14 and 5/22/17. Seven others were injected via their ventriculo-peritoneal shunts. Ten patients had Alzheimer's disease (AD), 6 had amyotrophic lateral sclerosis (ALS), 6 had progressive multiple sclerosis (MS-P), 6 had Parkinson's "Plus" (PD+), 1 had spinal cord injury, 1 had traumatic brain injury, and 1 had stroke. Median age was 74 (range 41-83). Injections were planned every 2-3 months. Thirty-one patients had 113 injections. Patients received SVF injection volumes of 3.5-20 cc (median:4 cc) containing 4.05 × 105 to 6.2 × 107 cells/cc, which contained an average of 8% hematopoietic and 7.5% adipose stem cells. Follow-up ranged from 0 to 36 months (median: 9.2 months). MRIs post injection(s) were unchanged, except for one AD patient whose hippocampal volume increased from < 5th percentile to 48th percentile (NeuroQuant® volumetric MRI). Of the 10 AD patients, 8 were stable or improved in tests of cognition. Two showed improvement in P-tau and ß-amyloid levels. Of the 6 MS-P patients all are stable or improved. Four of 6 ALS patients died of disease progression. Twelve of 111 injections (11%) led to 1-4 days of transient meningismus, and mild temperature elevation, which resolved with acetaminophen and/or dexamethasone. Two (1.8% of injections) required hospitalization for these symptoms. One patient (0.9% of injections) had his reservoir removed and later replaced for presumed infection. In this Phase 1 safety trial, ADSVF was safely injected into the human brain ventricular system in patients with no other treatment options. Secondary endpoints of clinical improvement or stability were particularly promising in the AD and MS-P groups. These results will be submitted for a Phase 2 FDA-approved trial.

Personal cell therapy for interstitial cystitis with autologous stromal vascular fraction stem cells.

BACKGROUND: The objective of this study was to evaluate whether autologous stem-cell-based therapy may mitigate the symptoms of interstitial cystitis. METHODS: Stromal vascular fraction (SVF) rich in stem cells and derived from autologous adipose tissue was deployed into 109 men and women with interstitial cystitis/painful bladder syndrome as a surgical procedure. This stem-cell-rich biologic product was injected both systemically and regionally into pelvic floor targets. Patients were queried about quality of life and symptom and bother subjective outcomes tests every 3 months for 2 years. RESULTS: A total of 78 patients reported a positive response at 1 year. Symptom and bother metrics were statistically improved at 1 year. There were minimal adverse events associated with the harvesting, procurement, and clinical deployment of SVF. CONCLUSION: Interstitial cystitis is a complex clinical problem that is known for its resistance to conventional therapies. SVF as an autologous personalized regenerative strategy shows good safety and efficacy and may potentially have a role in the mitigation of interstitial cystitis.

Effective Treatment of Traumatic Brain Injury in Rowett Nude Rats with Stromal Vascular Fraction Transplantation.

Traumatic brain injury (TBI) affects 1.9 million Americans, including blast TBI that is the signature injury of the Iraq and Afghanistan wars. Our project investigated whether stromal vascular fraction (SVF) can assist in post-TBI recovery. We utilized strong acoustic waves (5.0 bar) to induce TBI in the cortex of adult Rowett Nude (RNU) rats. One hour post-TBI, harvested human SVF (500,000 cells suspended in 0.5 mL lactated Ringers) was incubated with Q-Tracker cell label and administered into tail veins of RNU rats. For comparison, we utilized rats that received SVF 72 h post-TBI, and a control group that received lactated Ringers solution. Rotarod and water maze assays were used to monitor motor coordination and spatial memories. Rats treated immediately after TBI showed no signs of motor skills and memory regression. SVF treatment 72 h post-TBI enabled the rats maintain their motor skills, while controls treated with lactated Ringers were 25% worse statistically in both assays. Histological analysis showed the presence of Q-dot labeled human cells near the infarct in both SVF treatment groups; however, labeled cells were twice as numerous in the one hour group. Our study suggests that immediate treatment with SVF would serve as potential therapeutic agents in TBI.

The ratio of ADSCs to HSC-progenitors in adipose tissue derived SVF may provide the key to predict the outcome of stem-cell therapy.

BACKGROUND: Stromal vascular fraction (SVF) represents an attractive source of adult stem cells and progenitors, holding great promise for numerous cell therapy approaches. In 2017, it was reported that 1524 patients received autologous SVF following the enzymatic digestion of liposuction fat. The treatment was safe and effective and patients showed significant clinical improvement. In a collaborative study, we analyzed SVF obtained from 58 patients having degenerative, inflammatory, autoimmune diseases, and advanced stage cancer. RESULTS: Flow analysis showed that freshly isolated SVF was very heterogeneous and harbored four major subsets specific to adipose tissue; CD34high CD45- CD31- CD146- adipose-derived stromal/stem cells (ADSCs), CD34low CD45+ CD206+CD31- CD146- hematopoietic stem cell-progenitors (HSC-progenitors), CD34high CD45- CD31+CD146+ adipose tissue-endothelial cells and CD45-CD34-CD31-CD146+ pericytes. Culturing and expanding of SVF revealed a homogenous population lacking hematopoietic lineage markers CD45 and CD34, but were positive for CD90, CD73, CD105, and CD44. Flow cytometry sorting of viable individual subpopulations revealed that ADSCs had the capacity to grow in adherent culture. The identity of the expanded cells as mesenchymal stem cells (MSCs) was further confirmed based on their differentiation into adipogenic and osteogenic lineages. To identify the potential factors, which may determine the beneficial outcome of treatment, we followed 44 patients post-SVF treatment. The gender, age, clinical condition, certain SVF-dose and route of injection, did not play a role on the clinical outcome. Interestingly, SVF yield seemed to be affected by patient's characteristic to various extents. Furthermore, the therapy with adipose-derived and expanded-mesenchymal stem cells (ADE-MSCs) on a limited number of patients, did not suggest increased efficacies compared to SVF treatment. Therefore, we tested the hypothesis that a certain combination, rather than individual subset of cells may play a role in determining the treatment efficacy and found that the combination of ADSCs to HSC-progenitor cells can be correlated with overall treatment efficacy. CONCLUSIONS: We found that a 2:1 ratio of ADSCs to HSC-progenitors seems to be the key for a successful cell therapy. These findings open the way to future rational design of new treatment regimens for individuals by adjusting the cell ratio before the treatment.

Validation of Acoustic Wave Induced Traumatic Brain Injury in Rats.

BACKGROUND: This study looked to validate the acoustic wave technology of the Storz-D-Actor that inflicted a consistent closed-head, traumatic brain injury (TBI) in rats. We studied a range of single pulse pressures administered to the rats and observed the resulting decline in motor skills and memory. Histology was observed to measure and confirm the injury insult. METHODS: Four different acoustic wave pressures were studied using a single pulse: 0, 3.4, 4.2 and 5.0 bar (n = 10 rats per treatment group). The pulse was administered to the left frontal cortex. Rotarod tests were used to monitor the rats' motor skills while the water maze test was used to monitor memory deficits. The rats were then sacrificed ten days post-treatment for histological analysis of TBI infarct size. RESULTS: The behavioral tests showed that acoustic wave technology administered an effective insult causing significant decreases in motor abilities and memory. Histology showed dose-dependent damage to the cortex infarct areas only. CONCLUSIONS: This study illustrates that the Storz D-Actor effectively induces a repeatable TBI infarct, avoiding the invasive procedure of a craniotomy often used in TBI research.

Stromal Vascular Fraction Combined with Shock Wave for the Treatment of Peyronie's Disease.

BACKGROUND: This pilot study was used to evaluate safety and subjective outcomes in a small series of Peyronie's disease patients using a combination of autologous stromal vascular fraction (SVF) and penile shock wave treatments. SVF can be procured and deployed into Peyronie's plaques, enabling the surgeons to procure and mobilize significant numbers of both adult mesenchymal stem cells and antiinflammatory cytokines released from the adipose collagen matrix after collagen digestion. Penile shock wave therapy stimulates targeted tissues and may activate stem cells found in the SVF and promote healing and fibrosis mitigation. METHODS: SVF isolated from lipoaspirate was deployed by injection into 11 patients with Peyronie's plaques in combination with a series of shock wave treatments. Subjective outcomes tests performed at baseline and at 6 months included the Erectile Hardness Grading Score and the Peyronie's Disease Questionnaire (Questions 1-6). RESULTS: All patients noted subjective improvement in curvature and subjective reduction in plaque size. Seven patients reported improvement in erectile function. Mean Erectile Hardness Grading Score increased from 2.7 to 3.5, and mean Peyronie's Disease Questionnaire scores decreased from 15.0 to 8.7. CONCLUSIONS: SVF is known to have scar mitigation, antiinflammatory, immunomodulatory, and regenerative effects, and it has been used for a variety of conditions on an investigational basis. SVF containing mesenchymal stem cells can be procured in a closed surgical system from lipoaspirate in a same-day setting and deployed directly into Peyronie's plaques in combination with penile shock wave therapy resulting in plaque mitigation.

Intravesical instillation of pentosan polysulfate encapsulated in a liposome nanocarrier for interstitial cystitis.

We determined the effect of intravesical instillation of pentosan polysulfate encapsulated in liposomes for refractory interstitial cystitis patients. This was an open label uncontrolled study. Subjects were recruited from a private urology practice. Inclusion criteria included patients who met NIDDK criteria for Interstitial Cystitis (IC) and who were responding poorly to conventional treatments. Exclusion criteria included evidence of a urinary tract infection, bladder cancer, or other forms of chronic cystitis. Patients received 400 mg of Pentosan Polysulfate (PP) encapsulated into liposomes as an intravesical instillation performed every 2 weeks for 3 months. Baseline and post treatment outcome measures were obtained that included the O'Leary-Sant Interstitial Cystitis Symptom and Problem Questionnaire and the Pelvic Pain and Urgency/Frequency Patient symptom Scale tests. A total of 37 instillations were used and no adverse events occurred. Clinically significant decreases in symptom scores greater than 50% were seen in virtually all outcome measures at 3 month follow up. All subjects reported remarkable subjective improvement in pain symptoms marked by decreased use of narcotics and increased enjoyment of daily activities. No patients developed systemic symptoms or poor tolerance of the instillations. Intravesical Pentosan Polysulfate encapsulated into liposomes can significantly decrease frequency, urgency, pain and improve quality of life for two months after deployment. Additional studies are needed to determine cellular effects of glycosaminoglycan restoration, ideal doses, dosing intervals, safety and cost-effectiveness of this therapy.