Dietz, Allan B

Biliary complications (strictures and leaks) represent major limitations in living donor liver transplantation. Mesenchymal stem cells (MSCs) are a promising modality to prevent biliary complications because of immunosuppressive and angiogenic properties. Our goal was to evaluate the safety of adipose‐derived MSC delivery to biliary anastomoses in a porcine model. Secondary objectives were defining the optimal method of delivery (intraluminal versus extraluminal) and to investigate MSC engraftment, angiogenesis, and fibrosis. Pigs were divided into 3 groups. Animals underwent adipose collection, MSC isolation, and expansion. Two weeks later, animals underwent bile duct transection, reanastomosis, and stent insertion. Group 1 received plastic stents wrapped in unseeded Vicryl mesh. Group 2 received stents wrapped in MSC‐seeded mesh. Group 3 received unwrapped stents with the anastomosis immersed in an MSC suspension. Animals were killed 1 month after stent insertion when cholangiograms and biliary tissue were obtained. Serum was collected for liver biochemistries. Tissue was used for hematoxylin‐eosin and trichrome staining and immunohistochemistry for MSC markers (CD44 and CD34) and for a marker of neoangiogenesis (CD31). There were no intraoperative complications. One pig died on postoperative day 3 due to acute cholangitis. All others recovered without complications. Cholangiography demonstrated no biliary leaks and minimal luminal narrowing. Surviving animals exhibited no symptoms, abnormal liver biochemistries, or clinically significant biliary stricturing. Group 3 showed significantly greater CD44 and CD34 staining, indicating MSC engraftment. Fibrosis was reduced at the anastomotic site in group 3 based on trichrome stain. CD31 staining of group 3 was more pronounced, supporting enhanced neoangiogenesis. In conclusion, adipose‐derived MSCs were safely applied to biliary anastomoses. MSCs were locally engrafted within the bile duct and may have beneficial effects in terms of fibrosis and angiogenesis.

The interest in the use of human dendritic cells in cancer immunotherapy calls for efficient ex vivo methods of dendritic cell education. To extend the range of methods available, we generated phenotypically characteristic dendritic cells from peripheral blood monocytes incubated with granulocyte-macrophage colony-stimulating factor and interleukin-4 and infected them with an adenovirus containing a humanized version of green fluorescent protein as a marker of gene expression. The levels of expressed protein were high, but they were further increased in combination with cationic liposomes. In comparison to transfection efficiency of the homologous expression plasmid, adenovirus-mediated gene transfer was substantially more efficient. With the aid of liposome-mediated infection, gene transfer into CD83+ dendritic cells was highly effective, resulting in more than 90% of the cells expressing the transgene.

Abstract In multiple myeloma, the neoplastic plasma cells are either dispersed throughout the red marrow spaces of the axial and proximal appendicular skeleton or concentrated in discrete, well vascularlized plasmacytomas that stimulate osteoclastic bone resorption and erode into cortical bone. Interactions between bone marrow stromal cells (BMSC) and myeloma cells are clearly important in the pathogenesis of myeloma that diffusely infiltrates the bone marrow and where plasmacytomas are in contact with the bone marrow space. However, these interactions cannot be of major relevance at the centers of expanding plasmacytomas since these sites are devoid of osteoclasts, osteoblasts and other BMSC. Histological sections of skeletal plasmacytomas from 22 patients with multiple myeloma were therefore examined after immunostaining with anti-macrophage antibodies. Hematoxylin and eosin stained sections showed sheets of plasma cells interspersed with numerous small blood vessels. Immunostained sections revealed abundant CD68+, CD163+ macrophage infiltrates in all tumors, and scattered collections of CD3+ T lymphocytes. Tumor-associated macrophages (TAM) accounted for 2 to 12 percent of nucleated cells in these plasmacytomas and were evenly distributed through the parenchyma or clustered around CD34+ blood vessels. In general the TAM had dendritic morphology, and each dendrite was in close contact with multiple plasma cells. In a small percentage of cases, there was a subpopulation of TAM strikingly clustered around CD34+ positive blood vessels. However, vascular mimicry was not seen. Abundant murine CD68+ TAM were also found in subcutaneous KAS6/1 and MM1 myeloma xenografts in SCID mice. To determine whether cells of the monocytic lineage might be exploitable as carriers for systemic delivery of therapeutic agents to plasmacytomas, monocyte-derived dendritic cells were infected with oncolytic measles viruses and administered intravenously to SCID mice bearing subcutaneous KAS6/1 xenografts. Immunostaining for human MHC II antigen revealed that the infused DCs could localize to the KAS6/1 tumors where they heterofused with and transferred MV infection to the myeloma cells. This is the first report of TAM in skeletal plasmacytomas of myeloma patients. Further research into the role of TAM in supporting myeloma growth is warranted since they may offer many potential opportunities for the development of novel approaches to myeloma therapy.

Abstract Background Crohn’s rectovaginal fistulizing disease remains notoriously difficult to treat. A phase I clinical trial to evaluate the safety and feasibility of a novel protocol using a mesenchymal stem cell (MSC)–coated Gore Bio-A fistula plug for the treatment of medically and surgically refractory Crohn’s rectovaginal fistulas was conducted. Methods Five patients underwent an autologous subcutaneous adipose tissue harvest via a 2-cm abdominal wall incision at time of exam under anesthesia (EUA) with seton placement. MSCs were isolated, expanded, and impregnated on the plug. After 6 weeks, patients returned to the operating room for placement of the MSC-coated plug. The primary end points were safety and feasibility; the secondary end point was clinical and radiographic healing at 6 months. Results Five female patients (median age [range], 49 [38–53] years) with a median disease duration (range) of 23 (7–34) years who were on biologic (n = 5) or combination therapy (n = 3) had successful harvest and expansion of MSCs and delivery of the Gore Bio-A plug. There were no serious adverse events or adverse events related to the MSCs or plug during the 6-month follow-up. At 6 months, 3 patients had complete cessation of drainage, and 2 had >50% reduction in drainage; all had a persistent fistula tract identified on magnetic resonance imaging and EUA at 6 months. Conclusions Surgical placement of an autologous adipose-derived MSC-coated fistula plug in diverted patients with Crohn’s rectovaginal fistulas was safe and feasible. All patients had a reduction in the size of their fistula tract, and 3 of 5 had cessation of drainage, but none achieved complete healing. This was a phase I clinical trial of autologous mesenchymal stem cells on a plug for rectovaginal Crohn’s fistulas.

Abstract Background Immune editing is a major mechanism used by tumors to promote their survival. We have previously reported the presence of immunosuppressive monocytes (CD14+HLA-DR low/neg) in a number of cancers. Increased presence of these cells was associated with decreased treatment response and OS. Here we report an in vitro model to examine tumor monocyte cross talk and demonstrate that these monocytes may directly promote tumor cells’ resistance to chemotherapy. Method Monocytes from healthy donors were co-cultured with lymphoma cell lines (OCI-Ly1, OCI-Ly3, OCI-Ly7, OCI-Ly10, Su-DHL2, Su-DHL6, Jeko-1, Granta-519) with or without doxorubicin (DOX). Cultured cells were assessed for phenotype, viability, proliferation, and induced protein expression. Changes in vitro were confirmed in primary tissue by gene expression profile from 48 primary diffuse large B cell (DLBCL) lymphoma tumors. Results Monocytes converted CD14+HLA-DR+ to the CD14+HLA-DRlow/neg phenotype by co-culture with 5 of the 8 B cell lymphoma cell lines. The HLA-DR suppression was not due to the known regulatory effects of IL-10 nor was the effect seen in co-culture using B cells from healthy donors. Thus, the magnitude of DR loss is mediated by a yet undetermined mechanism. DOX incubation induced apoptosis in all of the cell lines. Co-culture with monocytes improved lymphoma cell survival in 3 of the 4 lymphoma cell lines. For example, untreated OCI-Ly3 had a 1.3±0.2 fold expansion that was reduced to 0.6±0.1 when treated with DOX (p<0.0001, n=13). However, when the cells were treated with DOX and co-cultured with monocytes the survival improved to 0.8±0.2 (p<0.0001, n=13). Having previously shown that CD14+HLA-DRlow/neg were capable of suppressing patient immunity, partially through arginase I, we determined if co-culture of monocytes with tumor cells increased arginase I expression in either monocytes or tumor cells. Not only did we see arginase I expression increases during co-culture(n=10, p<0.01), but surprisingly, the greatest increases were seen during co-culture and DOX treatment (n=10, p=0.01). To determine if our in vitro observations reflect human tumors, we used gene expression data obtained from 48 DLBCL tumors and found that increased CD14 gene expression was positively correlated with increased arginase I expression (n=48, p=0.02). Conclusions We have demonstrated that certain tumor cells induced phenotype changes in monocytes in an IL-10 independent and a tumor specific way. In those cells where tumors factors mediate monocyte phenotype, the monocytes can promote tumor cell resistance to cytotoxic killing by DOX, thus demonstrating a cross-talk between monocyte and tumor function. We found that in an environment of monocyte/tumor cross talk, arginase I expression is further increased in a cytotoxic environment. Together, this data demonstrates an active cross talk between monocytes and tumors resulting in multiple mechanisms of tumor resistance to chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Background: The lack of a standardized system for describing cell therapies acts as a barrier to advancement in clinical and basic research and practice. The aim of this study was to establish an international expert consensus on strategies to improve standardization and transparency when describing cell therapies. The secondary aim was to develop a consensus among experts on the contents of a standardized tool for describing cell therapies. Methods: The need for expert consensus on strategies to improve cell therapy communication was confirmed at the American Academy of Orthopaedic Surgeons/National Institutes of Health Optimizing Clinical Use of Biologics Symposium in 2018. A working group of 6 experts convened an international consensus process involving clinicians and basic scientists using validated Delphi methodology. This iterative process was used to define statements on communication of cell therapies and develop a standardized tool for describing cell therapies. Results: Thirty-four experts completed 3 rounds survey with use of the Delphi process. After 3 rounds, 27 statements relating to existing nomenclature, solutions to improve communication, ideal characteristics of a framework, mandatory elements of a new framework, and future work to facilitate application reached consensus with >80% agreement and <5% disagreement. Consensus was reached on the contents of a tool for improving standardization and transparency when describing cell therapies. This tool, dubbed “DOSES,” is based on the reporting of 5 core items: donor (i.e., autologous, allogeneic, xenogeneic), origin of tissue, separation from other cell types/preparation method, exhibited cell characteristics associated with behavior, and the site of delivery. Conclusions: This study has established expert consensus on the communication of cell therapies. The DOSES tool has been developed to improve standardization and transparency in describing cell therapies. Clinical Relevance: The DOSES tool for describing cell therapies can be utilized by researchers, clinicians, regulators, and industry professionals to improve standardization and transparency when describing cell therapies. The use of this tool may allow clinicians and patients to better understand the characteristics of current and future cell preparations.

ObjectiveTo assess the biologic effects of lidocaine on the viability, proliferation, and function of human adipose tissue–derived mesenchymal stromal/stem cells (MSCs) in vitro.MethodsAdipose‐derived MSCs from three donors were exposed to lidocaine at various dilutions (2 mg/mL to 8 mg/mL) and exposure times (0.5 to 4 hours). Cell number and viability, mitochondrial activity, and real‐time reverse‐transcriptase quantitative polymerase chain reaction (RT‐qPCR) were analyzed at 0 (immediate effects) or 24 and 48 hours (recovery effects) after treatment with lidocaine.ResultsTrypan blue staining showed that increasing concentrations of lidocaine decreased the number of observable viable cells. 3‐[4,5,dimethylthiazol‐2‐yl]‐5‐[3‐carboxymethoxy‐phenyl]‐2‐[4‐sulfophenyl]‐2H‐tetrazolium (MTS) assays revealed a concentration‐ and time‐ dependent decline of mitochondrial activity and proliferative ability. Gene expression analysis by RT‐qPCR revealed that adipose‐derived MSCs exposed to lidocaine express robust levels of stress response/cytoprotective genes. However, higher concentrations of lidocaine caused a significant downregulation of these genes. No significant differences were observed in expression of extracellular matrix (ECM) markers COL1A1 and DCN except for COL3A1 (P < .05). Levels of messenger RNA (mRNA) for proliferation markers (CCNB2, HIST2H4A, P < .001) and MKI67 (P < .001) increased at 24 and 48 hours. Expression levels of several transcription factors— including SP1, PRRX1, and ATF1—were modulated in the same manner. MSC surface markers CD44 and CD105 demonstrated decreased expression immediately after treatment, but at 24 and 48 hours postexposure, the MSC markers showed no significant difference among groups.ConclusionLidocaine is toxic to MSCs in a dose‐ and time‐ dependent manner. MSC exposure to high (4‐8 mg/mL) concentrations of lidocaine for prolonged periods can affect their biologic functions. Although the exposure time in vivo is short, it is essential to choose safe concentrations when applying lidocaine along with MSCs to avoid compromising the viability and potency of the stem cell therapy.

BACKGROUND: Management of transsphincteric cryptoglandular fistulas remains a challenging problem and the optimal surgical approach remains elusive. Mesenchymal stem cells, increasingly being utilized for perianal Crohn’s disease, offer a novel therapy to treat cryptoglandular fistulas. OBJECTIVES: This study aimed to determine safety and feasibility of using an autologous mesenchymal stem cell-coated fistula plug in patients with transsphincteric cryptoglandular fistulas. DESIGN: This study is a phase I clinical trial. SETTING: This study was conducted at a tertiary academic medical center. PATIENTS: Adult (>18 years) male and female patients with transsphincteric cryptoglandular fistulas were selected. MAIN OUTCOMES MEASURES: The primary outcomes measured were the safety, feasibility, and efficacy of a mesenchymal stem cell-coated fistula plug in patients with transsphincteric fistulas. RESULTS: Fifteen patients (8 women, mean age 39.8 years) with a single-tract transsphincteric fistula received a mesenchymal stem cell-loaded fistula plug and were followed for 6 months. Duration of disease at the time of study enrollment was a median of 3.0 years (range, 1–13 years) with a median of 3.5 (range, 1–20) prior surgical interventions. Adverse events included 1 plug extrusion, 1 abdominal wall seroma, 3 perianal abscesses requiring drainage, and 1 patient with perianal cellulitis. There were no serious adverse events. At 6 months, 3 patients had complete clinical healing, 8 had partial healing, and 4 patients showed no clinical improvement. Radiographic improvement was seen in 11 of 15 patients. LIMITATIONS: This study was limited by the small cohort and short follow-up. CONCLUSIONS: Autologous mesenchymal stem cell-coated fistula plug treatment of transsphincteric cryptoglandular fistulas was safe and feasible and resulted in complete or partial healing in a majority of patients. See Video Abstract at http://links.lww.com/DCR/A897.