Lisa Stehno-Bittel

Introduction: Multipotent Stromal Cells (MSCs) are utilized as therapeutic agents for addressing musculoskeletal conditions, including knee osteoarthritis (OA). However, major challenges in the clinical application include maintenance of the cells in the joint capsule. Hyaluronic acid (HA) is endogenous in synovial joints and commercially available as a joint lubricant. We tested the hypothesis that delivery of MSCs in HA into an OA rat knee model could improve outcomes. Methods: Rat bone marrow MSCs were suspended in a commercially available HA paste, and cell viability measured with live/dead stains. Biomarkers for MSC chondrogenesis and osteogenesis were monitored with PCR. MSCs with or without HA were injected into the knees of OA rats and histology conducted 6 weeks later. Results: Suspending MSC in HA resulted in a slight reduction in viability. The gene expression profile showed an increase in MSC biomarkers for cells in HA with a decrease in osteogenic markers. Four groups of treatment (vehicle, MSCs alone, HA alone, MSCs + HA) were injected into the knees of osteoarthritic rats. Pain scores, collected weekly, showed no difference between the groups. Immunohistochemistry for inflammatory markers illustrated no obvious differences between groups. Proteoglycans, indicative of cartilage, showed a loss in the vehicle group and modest signs of cartilage with MSCs alone, but when mixed with the HA, any benefit was lost. OARSI Histological Scoring completed by 2 independent technicians concluded no improvement in joint integrity with the addition of HA. Conclusion: A commercially available HA failed to enhance joint regeneration compared to MSCs alone.

Background: Protection of islets without systemic immunosuppression has been a long-sought goal in the islet transplant field. We conducted a pilot biocompatibility/safety study in healthy dogs followed by a dose-finding efficacy study in diabetic dogs using polyethylene glycol diacrylate (PEGDA) microencapsulated allogeneic canine islets.Methods: Prior to the transplants, characterization of the canine islets included the calculations determining the average cell number/islet equivalent. Following measurements of purity, insulin secretion, and insulin, DNA and ATP content, the islets were encapsulated and transplanted interperitoneally into dogs via a catheter, which predominantly attached to the omentum. In the healthy dogs, half of the microspheres injected contained canine islets, the other half of the omentum received empty PEGDA microspheres.Results: In the biocompatibility study, healthy dogs received increasing doses of cells up to 1.7 M cells/kg body weight, yet no hypoglycemic events were recorded and the dogs presented with no adverse events. At necropsy the microspheres were identified and described as clear with attachment to the omentum. Several of the blood chemistry values that were abnormal prior to the transplants normalized after the transplant. The same observation was made for the diabetic dogs that received higher doses of canine islets. In all diabetic dogs, the insulin required to attempt to control blood glucose was cut by 50–100% after the transplant, down to no required insulin for the course of the 60-day study. The dogs had no adverse events and behavioral monitoring suggested normal activity after recovery from the transplant.Conclusions and implications: The study provides evidence that PEGDA microencapsulated canine islets reversed the signs of diabetes without immunosuppression and led to states of insulin-independence or significantly lowered insulin requirements in the recipients.

Background: The source of multipotent stromal cells (MSC) can have a significant influence on the health and expansion capacity of the cells. As the applications for allogeneic MSCs in the treatment of feline diseases increase, the location of the initial donor tissue must be analyzed. To date, comparisons have only been made between feline MSCs collected from bone marrow or abdominal fat. This is the first report to compare cells obtained from different adipose depots in the cat with a focus on clinically relevant donor tissues. The tissue was collected from 34 healthy cats undergoing spaying (fat around the ovaries and uterine horn) or subcutaneous fat collected during surgical procedures. Results: The amount of starting material is essential to isolate sufficient MSCs. The total tissue yield from the subcutaneous fat was significantly greater than could be obtained from around the reproductive organs, leading to 3 times more MSCs per donor. However, the concentration of MSCs obtained from reproductive fat was higher than from subcutaneous fat. In addition, the viability of the MSCs from the reproductive fat was significantly higher than the subcutaneous fat. Since most spaying occurs in young cats (under 18 months) reproductive fat was collected from adult cats during spaying, illustrating that age did not alter the yield or viability of the MSCs. When sufficient tissue was collected, it was digested either mechanically or enzymatically. Mechanical digestion further decreased the viability and yield of MSCs from subcutaneous fat compared to enzymatic digestion. Biomarkers of stem cell characterization, expansion capacity and function were detected using qPCR. CD70, CD90 and CD105 were all expressed in high levels in the 3 groups. However, the reproductive fat had higher levels of CD73 with the mechanically digested subcutaneous fat having the least. Gata6 was detected in all samples while Sox2 and Sox17 were also detected with higher quantities found in the enzymatically digested subcutaneous fat. Negative control genes of Gata4 and Pdx1 showed no detection prior to 50 cycles. During the first three passages, age of the donor, location of the donor tissue, or digestion protocol had no effect on cell culture doubling times or cell viability. Conclusions: While MSCs from reproductive fat had superior cells/tissue weight and initial viability, there were still dramatically fewer cells obtained compared to subcutaneous fat due to the limited amount of tissue surrounding the reproductive organs. Further, in P1-P3 cultures there were no differences noted in doubling time or cell viability between tissue obtained from reproductive or subcutaneous fat depots.

Background: The source of multipotent stromal cells (MSC) can have a significant influence on the health and expansion capacity of the cells. As the applications for allogeneic MSCs in the treatment of feline diseases increase, the location of the initial donor tissue must be analyzed. To date, comparisons have only been made between feline MSCs collected from bone marrow or abdominal fat. This is the first report to compare cells obtained from different adipose depots in the cat. The adipose tissue was collected from 32 healthy cats undergoing spaying (fat around the ovaries and uterine horn) or subcutaneous fat collected during surgical procedures. Results: The total tissue yield from the subcutaneous fat was significantly greater than could be obtained from around the reproductive organs, leading to 3 times more total MSCs. However, the density of MSCs obtained from reproductive fat was significantly higher than from subcutaneous fat. In addition, the viability of the MSCs from the reproductive fat was significantly higher than the subcutaneous fat. When sufficient tissue was collected, it was digested either mechanically or enzymatically. Mechanical digestion further decreased the viability and yield of MSCs from subcutaneous fat compared to enzymatic digestion. Biomarkers of stem cell expansion and function were detected using qPCR. Gata6 was detected in all samples similarly regardless of site of harvest or method of digestion. Sox2 and Sox17 were also detected in all samples with higher quantities found in the enzymatically digested subcutaneous fat. Control genes of Gata4 and Pdx1 were detected in very low levels. Negative controls showed no detection prior to 50 cycles. During the first passage there was no statistically significant difference in doubling times or viability. But at P2, MSCs from the enzymatically-digested reproductive fat had the lowest doubling time and a trend towards the highest viability. Conclusion: Feline reproductive adipose tissue is a reasonable source of MSCs for eventual therapeutic applications with superior cell density, viability and expansion

The field of cell therapy has blossomed, providing exciting new options for treating a variety of diseases. While few cell therapy products have US FDA approval, there are thousands of cell treatme...

Cancer is the second leading cause of death in the United States, with more than 1 million new cancer cases diagnosed each year. Yet, survival from cancer has been increasing dramatically, with more than 2 million fewer cancer deaths during the past 2 decades than in previous decades. Physical therapists are familiar with the side effects of common chemotherapies and radiation therapy, but new immunotherapy drugs coming to the market have the potential to completely change the cancer treatment landscape. They provide new hope for cures that previously were not possible, but they also have their own side effects and toxicity issues. Because of their recent introduction to the market, no studies have examined the effects of immunotherapies on cancer rehabilitation, yet it is an essential question. The purpose of this article is to review 2 categories of new cancer immunotherapy treatments: checkpoint inhibitors and chimeric antigen receptor T cells. The physiological mechanism, known side effects, and toxicities are reviewed. We discuss the implications for physical therapists caring for cancer survivors and propose conservative interventions, ensuring that therapists provide the highest level of care for our patients with cancer. The purpose of this article is to inform the rehabilitation professional and set the baseline understanding for subsequent research studies elucidating the long-term effect of immunotherapies on cancer rehabilitation.

Alginate has long been the material of choice for immunoprotection of islets due to its low cost and ability to easily form microspheres. Unfortunately, this seaweed-derived material is notoriously prone to fibrotic overgrowth in vivo, resulting in premature graft failure. The purpose of this study was to test an alternative, hyaluronic acid (HA-COL), for in vitro function, viability, and allogeneic islet transplant outcomes in diabetic rats. In vitro studies indicated that the HA-COL gel had diffusion characteristics that would allow small molecules such as glucose and insulin to enter and exit the gel, whereas larger molecules (70 and 500 kDa dextrans) were impeded from diffusing past the gel edge in 24 h. Islets encapsulated in HA-COL hydrogel showed significantly improved in vitro viability over unencapsulated islets and retained their morphology and glucose sensitivity for 28 days. When unencapsulated allogeneic islet transplants were administered to the omentum of outbred rats, they initially were normoglycemic, but by 11 days returned to hyperglycemia. Immunohistological examination of the grafts and surrounding tissue indicated strong graft rejection. By comparison, when using the same outbred strain of rats, allogeneic transplantation of islets within the HA-COL gel reversed long-term diabetes and prevented graft rejection in all animals. Animals were sacrificed at 40, 52, 64, and 80 weeks for evaluation, and all were non-diabetic at sacrifice. Explanted grafts revealed viable islets in the transplant site as well as intact hydrogel, with little or no evidence of fibrotic overgrowth or cellular rejection. The results of these studies demonstrate great potential for HA-COL hydrogel as an alternative to sodium alginate for long-term immunoprotected islet transplantation.

Canine diabetes is a strikingly prevalent and growing disease, and yet the standard treatment of a twice-daily insulin injection is both cumbersome to pet owners and only moderately effective. Islet transplantation has been performed with repeated success in canine research models, but has unfortunately not been made available to companion animals. Standard protocols for islet isolation, developed primarily for human islet transplantation, include beating-heart organ donation, vascular perfusion of preservation solutions, specialized equipment. Unfortunately, these processes are prohibitively complex and expensive for veterinary use. The aim of the study was to develop a simplified approach for isolating canine islets that is compatible with the financial and logistical restrictions inherent to veterinary medicine for the purpose of translating islet transplantation to a clinical treatment for canine diabetes. Here, we describe simplified strategies for isolating quality islets from deceased canine donors without vascular preservation and with up to 90 min of cold ischemia time. An average of more than 1500 islet equivalents per kg of donor bodyweight was obtained with a purity of 70% (N = 6 animals). Islets were 95% viable and responsive to glucose stimulation for a week. We found that processing only the body and tail of the pancreas increased isolation efficiency without sacrificing islet total yield. Islet yield per gram of tissue increased from 773 to 1868 islet equivalents when the head of the pancreas was discarded (N = 3/group). In summary, this study resulted in the development of an efficient and readily accessible method for obtaining viable and functional canine islets from deceased donors. These strategies provide an ethical means for obtaining donor islets.

Pancreatic islets, especially the large islets (> 150µm in diameter) have poor survival rates in culture. Co-culturing with mesenchymal stem cells (MSCs) has been shown to improve islet survival and function. However, most co-culture studies have been comprised of MSC surrounding islets in the media. The purpose of this study was to determine whether islet survival and function was improved when the 2 populations of cells were intermingled with each other in a defined geometry. Hybrid spheroids containing 25, 50 or 75 or 90% islets cells with appropriate numbers of MSCs were created along with spheroids comprised of only islet cells or only MSCs. Spheroids were tested for yield, viability, diameter, cellular composition, and glucose-stimulated insulin secretion. The 25% islet/75% MSC group created the fewest spheroids, with the poorest survival and insulin secretion and the largest diameter. The remaining groups were highly viable with average diameters under 80µm at formation. However, the hybrid spheroid groups preferred to cluster in islet-only spheroids. The 50, 75 and 90% islet cell groups had excellent long-term survival with 90–95% viability at 2 weeks in culture, compared with the islet only group that were below 80% viability. The glucose-stimulated insulin secretion was not statistically different for the 50, 75, or 90 groups when exposed to 2.4, 16.8, or 22.4 mM glucose. Only the spheroids with 25% islet cells had a statistically lower levels of insulin release, and the 100% had statistically higher levels at 22.4 mM glucose and in response to secretagogue. Thus, imbedded co-culture improved long-term viability, but failed to enhance glucose-stimulated insulin secretion in vitro.