Optimized peripheral blood progenitor cell mobilization for autologous hematopoietic cell transplantation in children with high-risk and refractory malignancies.

BACKGROUND: Autologous hematopoietic stem cell transplantation (aHSCT) using hematopoietic progenitor cells (HPCs) has become an important therapeutic modality for patients with high-risk malignancies. Current literature on standardized method for HPC apheresis in children is sparse and failure rate reported as high as 30%. PATIENTS/METHODS: A retrospective study of 125 pediatric patients with high-risk malignancies undergoing aHSCT in Western Australia between 1997 and 2016 was conducted. RESULTS: Mobilization was achieved by means of chemotherapy and granulocyte colony-stimulating factor (G-CSF). Patients underwent apheresis the day after CD34+ counts reached ≥20/µL and an additional dose of G-CSF. Peripheral arterial and intravenous lines were inserted in pediatric intensive care unit under local anesthetic and/or sedation, omitting the need for general anesthesia as well as facilitating an uninterrupted apheresis flow. Larger apheresis total blood volumes were processed in patients weighing ≤20 kg. The minimal dose of ≥2 × 106 CD34+ cells/kg was successfully collected in 98.4% of all patients. The optimal dose of 3-5 × 106 CD34+ cells/kg was collected in 96% of patients scheduled for a single aHSCT, 87.5% for tandem, and 100% for triple aHSCT. All HPC collections were completed in one apheresis session. Mobilization after ≤3 chemotherapy cycles and cycles including cyclophosphamide resulted in a significantly higher yield of CD34+ cells. CONCLUSION: Our approach to HPC mobilization by means of chemotherapy and single myeloid growth factor combined with optimal collection timing facilitated by continuous apheresis flow resulted in highly effective HPC harvest in children and adolescents with high-risk cancers.

Cellular Immunotherapy for Hematologic Malignancies: Beyond Bone Marrow Transplantation.

Immunotherapy has changed treatment practices for many hematologic malignancies. Even in the current era of targeted therapy, chemotherapy remains the backbone of treatment for many hematologic malignancies, especially in acute leukemias, where relapse remains the major cause of mortality. Application of novel immunotherapies in hematology attempts to harness the killing power of the immune system against leukemia and lymphoma. Cellular immunotherapy is evolving rapidly for high-risk hematologic disorders. Recent advances include chimeric antigen-receptor T cells, mesenchymal stromal/stem cells, dendritic cell tumor vaccines, cytokine-induced killer cells, and virus-specific T cells. The advantages of nontransplantation cellular immunotherapy include suitability for patients for whom transplantation has failed or is contraindicated, and a potentially less-toxic treatment alternative to transplantation for relapsed/refractory patients. This review examines those emerging cellular immunotherapies that are changing treatment paradigms for patients with hematologic malignancies.

A phase 2 study of allogeneic mesenchymal stromal cells for luminal Crohn's disease refractory to biologic therapy.

BACKGROUND & AIMS: Transplantation of peripheral blood stem cells has been successful therapy for small numbers of patients with Crohn's disease (CD), but requires prior myeloconditioning. Mesenchymal stromal cells (MSCs) escape immune recognition, so myeloconditioning is not required before their administration. We investigated the efficacy of allogeneic MSCs in patients with luminal CD. METHODS: Our phase 2, open-label, multicenter study included 16 patients (21-55 y old; 6 men) with infliximab- or adalimumab-refractory, endoscopically confirmed, active luminal CD (CD activity index [CDAI], >250). Subjects were given intravenous infusions of allogeneic MSCs (2 × 10(6) cells/kg body weight) weekly for 4 weeks. The primary end point was clinical response (decrease in CDAI >100 points) 42 days after the first MSC administration; secondary end points were clinical remission (CDAI, <150), endoscopic improvement (a CD endoscopic index of severity [CDEIS] value, <3 or a decrease by >5), quality of life, level of C-reactive protein, and safety. RESULTS: Among the 15 patients who completed the study, the mean CDAI score was reduced from 370 (median, 327; range, 256-603) to 203 (median, 129) at day 42 (P < .0001). The mean CDAI scores decreased after each MSC infusion (370 before administration, 269 on day 7, 240 on day 14, 209 on day 21, 182 on day 28, and 203 on day 42). Twelve patients had a clinical response (80%; 95% confidence interval, 72%-88%; mean reduction in CDAI, 211; range 102-367), 8 had clinical remission (53%; range, 43%-64%; mean CDAI at day 42, 94; range, 44-130). Seven patients had endoscopic improvement (47%), for whom the mean CDEIS scores decreased from 21.5 (range, 3.3-33) to 11.0 (range, 0.3-18.5). One patient had a serious adverse event (2 dysplasia-associated lesions), but this probably was not caused by MSCs. CONCLUSIONS: In a phase 2 study, administration of allogeneic MSCs reduced CDAI and CDEIS scores in patients with luminal CD refractory to biologic therapy. ClinicalTrials.gov number, NCT01090817.

A phase II randomized vehicle-controlled trial of intradermal allogeneic fibroblasts for recessive dystrophic epidermolysis bullosa.

BACKGROUND: Chronic wounds are a major source of morbidity and mortality in generalized severe recessive dystrophic epidermolysis bullosa (RDEB-GS). OBJECTIVE: This was a phase II double-blinded randomized controlled trial of intralesional allogeneic cultured fibroblasts in suspension solution versus suspension solution alone for wound healing in RDEB-GS. METHODS: Adult patients with RDEB-GS were screened for chronic ulcers and reduced collagen VII expression. Up to 6 pairs of symmetric wounds were measured and biopsied at baseline, then randomized to cultured allogeneic fibroblasts in a crystalloid suspension solution with 2% albumin or suspension solution alone. Ulcer size, collagen VII protein and messenger RNA expression, anchoring fibril numbers, morphology, and inflammatory markers were measured at 2 weeks and at 3, 6, and 12 months. RESULTS: All wounds healed significantly more rapidly with fibroblasts and vehicle injections, with an area decrease of 50% by 12 weeks, compared with noninjected wounds. Collagen VII expression increased to a similar degree in both study arms in wounds from 3 of 5 patients. LIMITATIONS: The number of patients with RDEB-GS who met inclusion criteria was a limitation, as was 1 trial center rather than multicenter. CONCLUSIONS: The injection of both allogeneic fibroblasts and suspension solution alone improved wound healing in chronic nonhealing RDEB-GS wounds independently of collagen VII regeneration. This may provide feasible therapy for wound healing in patients with RDEB-GS.

Effects of amyloid precursor protein peptide APP96-110, alone or with human mesenchymal stromal cells, on recovery after spinal cord injury.

Delivery of a peptide (APP96-110), derived from amyloid precursor protein (APP), has been shown to elicit neuroprotective effects following cerebral stroke and traumatic brain injury. In this study, the effect of APP96-110 or a mutant version of this peptide (mAPP96-110) was assessed following moderate (200 kdyn, (2 N)) thoracic contusive spinal cord injury (SCI) in adult Nude rats. Animals received a single tail vein injection of APP96-110 or mAPP96-110 at 30 minutes post-SCI and were then assessed for functional improvements over the next 8 weeks. A cohort of animals also received transplants of either viable or non-viable human mesenchymal stromal cells (hMSCs) into the SC lesion site at one week post-injury to assess the effect of combining intravenous APP96-110 delivery with hMSC treatment. Rats were perfused 8 weeks post-SCI and longitudinal sections of spinal cord analyzed for a number of factors including hMSC viability, cyst size, axonal regrowth, glial reactivity and macrophage activation. Analysis of sensorimotor function revealed occasional significant differences between groups using Ladderwalk or Ratwalk tests, however there were no consistent improvements in functional outcome after any of the treatments. mAPP96-110 alone, and APP96-110 in combination with both viable and non-viable hMSCs significantly reduced cyst size compared to SCI alone. Combined treatments with donor hMSCs also significantly increased ßIII tubulin+, glial fibrillary acidic protein (GFAP+) and laminin+ expression, and decreased ED1+ expression in tissues. This preliminary study demonstrates that intravenous delivery of APP96-110 peptide has selective, modest neuroprotective effects following SCI, which may be enhanced when combined with hMSC transplantation. However, the effects are less pronounced and less consistent compared to the protective morphological and cognitive impact that this same peptide has on neuronal survival and behaviour after stroke and traumatic brain injury. Thus while the efficacy of a particular therapeutic approach in one CNS injury model may provide justification for its use in other neurotrauma models, similar outcomes may not necessarily occur and more targeted approaches suited to location and severity are required. All animal experiments were approved by The University of Western Australia Animal Ethics Committee (RA3/100/1460) on April 12, 2016.

Transcriptional profiling of circulating mononuclear cells from patients with chronic obstructive pulmonary disease receiving mesenchymal stromal cell infusions.

Chronic obstructive pulmonary disease (COPD) is an inflammatory airways disease with limited therapeutic options. We have previously shown that mesenchymal stromal cell (MSC) infusions are well tolerated in patients with COPD and reduce circulatory biomarkers associated with systemic inflammation and oxidative stress. This study aimed to delineate the underlying mechanisms further by characterizing the transcriptional networks in these patients and to explore the role of MSC-derived paracrine factors in regulating these pathways. Allogeneic, bone marrow-derived MSCs were systemically administered into patients with stable COPD (n = 9). Gene expression profiles from peripheral blood mononuclear cells (PBMCs) were analyzed across the first week after infusion. Paracrine mechanisms associated with these transcriptional changes were explored further by culturing patient PBMCs with MSC-conditioned medium (MSC-CM) or post-MSC infusion (PI) plasma to measure the regulatory effects of soluble factors that may be derived from MSCs. MSC-CM and PI-plasma were characterized further to identify potential immunoregulatory candidates. MSC infusion elicited a strong but transient transcriptional response in patient PBMCs that was sustained up to 7 days. MSC infusion strongly downregulated transcriptional pathways related to interleukin (IL)-8 and IL-1ß, which were also significantly inhibited in vitro following co-culture of PBMCs with MSC-CM and PI-plasma. MSC-derived soluble tumor necrosis factor receptor-1, transforming growth factor-ß1, and extracellular vesicle-associated microRNAs were identified as potential mechanisms promoting these changes, but depletion of these individual candidates revealed inconsistent results. MSC-derived paracrine factors modulate important inflammatory pathways that are relevant to COPD pathogenesis. These data strengthen the hypothesis that therapies using MSCs and their secreted products may be beneficial to patients with COPD.

Comparing the Effect of TGF-ß Receptor Inhibition on Human Perivascular Mesenchymal Stromal Cells Derived from Endometrium, Bone Marrow and Adipose Tissues.

Rare perivascular mesenchymal stromal cells (MSCs) with therapeutic properties have been identified in many tissues. Their rarity necessitates extensive in vitro expansion, resulting in spontaneous differentiation, cellular senescence and apoptosis, producing therapeutic products with variable quality and decreased potency. We previously demonstrated that A83-01, a transforming growth factor beta (TGF-ß) receptor inhibitor, maintained clonogenicity and promoted the potency of culture-expanded premenopausal endometrial MSCs using functional assays and whole-transcriptome sequencing. Here, we compared the effects of A83-01 on MSCs derived from postmenopausal endometrium, menstrual blood, placenta decidua-basalis, bone marrow and adipose tissue. Sushi-domain-containing-2 (SUSD2+) and CD34+CD31-CD45- MSCs were isolated. Expanded MSCs were cultured with or without A83-01 for 7 days and assessed for MSC properties. SUSD2 identified perivascular cells in the placental decidua-basalis, and their maternal origin was validated. A83-01 promoted MSC proliferation from all sources except bone marrow and only increased SUSD2 expression and prevented apoptosis in MSCs from endometrial-derived tissues. A83-01 only improved the cloning efficiency of postmenopausal endometrial MSCs (eMSCs), and expanded adipose tissue MSCs (adMSCs) underwent significant senescence, which was mitigated by A83-01. MSCs derived from bone marrow (bmMSCs) were highly apoptotic, but A83-01 was without effect. A83-01 maintained the function and phenotype in MSCs cultured from endometrial, but not other, tissues. Our results also demonstrated that cellular SUSD2 expression directly correlates with the functional phenotype.

Cranial reconstruction using allogeneic mesenchymal stromal cells: A phase 1 first-in-human trial.

Cranioplasty is necessary for patients that have undergone craniectomy following trauma, stroke or other causes of elevated intracranial pressure. This study assessed the effectiveness of treating cranial defects with allogeneic mesenchymal stromal cells (MSC) on a ceramic carrier and polymer scaffold, to produce viable bone and healing of a cranial void. Patients underwent a baseline computed tomography (CT) scan for construct design. Two sets of interlocking moulds were three-dimensional printed to enable shaping of two polymer meshes, which formed the boundaries of the construct corresponding to restoration of the skull interna and externa. In vitro expanded donor MSC were seeded onto ceramic granules in a good manufacturing practices facility. The inner mesh was placed in theatre, followed by the cell-loaded granules, and the outer mesh. Patients were followed-up at 3, 6 and 12 months and cosmesis assessed visually, while bone formation was assessed by CT scans at 1 day, 3 months and 12 months. Manufacture of the construct and surgery was uneventful for all three patients. Initial cosmesis was excellent with no complications. New bone formation was demonstrated by analysis of CT data; however, bone resorption was noted in all 3 cases on the 12-month CT scan. The lack of rigidity of the construct in an environment with continuous pulsatile movement may be preventing the formation of solid bone. It is possible to produce a customized allogeneic MSC construct for cranial reconstruction to replace cranial bone with good cosmesis, using a combination of medical computer modelling, rapid-prototyping and tissue engineering.

Mesenchymal Stromal Cell Therapy for Chronic Lung Allograft Dysfunction: Results of a First-in-Man Study.

Chronic lung transplant rejection (termed chronic lung allograft dysfunction [CLAD]) is the main impediment to long-term survival after lung transplantation. Bone marrow-derived mesenchymal stromal cells (MSCs) represent an attractive cell therapy in inflammatory diseases, including organ rejection, given their relative immune privilege and immunosuppressive and tolerogenic properties. Preclinical studies in models of obliterative bronchiolitis and human trials in graft versus host disease and renal transplantation suggest potential efficacy in CLAD. The purpose of this phase 1, single-arm study was to explore the feasibility and safety of intravenous delivery of allogeneic MSCs to patients with advanced CLAD. MSCs from unrelated donors were isolated from bone marrow, expanded and cryopreserved in a GMP-compliant facility. Patients had deteriorating CLAD and were bronchiolitis obliterans (BOS) grade ≥ 2 or grade 1 with risk factors for rapid progression. MSCs (2 x 106 cells per kilogram patient weight) were infused via a peripheral vein twice weekly for 2 weeks, with 52 weeks follow-up. Ten Patients (5 male, 8 bilateral, median [interquartile range] age 40 [30-59] years, 3 BOS2, 7 BOS3) participated. MSC treatment was well tolerated with all patients receiving the full dosing schedule without any procedure-related serious adverse events. The rate of decline in forced expiratory volume in one second slowed after the MSC infusions (120 ml/month preinfusion vs. 30 ml/month postinfusion, p = .08). Two patients died at 152 and 270 days post-MSC treatment, both from progressive CLAD. In conclusion, infusion of allogeneic bone marrow-derived MSCs is feasible and safe even in patients with advanced CLAD. Stem Cells Translational Medicine 2017;6:1152-1157.

Human mesenchymal stem cells attenuate early damage in a ventilated pig model of acute lung injury.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a major cause of global morbidity and mortality. Mesenchymal stem cells (MSC) have shown promise in treating inflammatory lung conditions. We hypothesised that human MSC (hMSC) can improve ALI/ARDS through their anti-inflammatory actions. We subjected pigs (n=6) to intravenous oleic acid (OA) injury, ventilation and hMSC infusion, while the controls (n=5) had intravenous OA, ventilation and an infusion vehicle control. hMSC were infused 1h after the administration of OA. The animals were monitored for additional 4h. Nuclear translocation of nuclear factor-light chain enhancer of activated B cells (NF-κB), a transcription factor that mediates several inflammatory pathways was reduced in hMSC treated pigs compared to controls (p=0.04). There was no significant difference in lung injury, assessed by histological scoring in hMSC treated pigs versus controls (p=0.063). There was no difference in neutrophil counts between hMSC-treated pigs and controls. Within 4h, there was no difference in the levels of IL-10 and IL-8 pre- and post-treatment with hMSC. In addition, there was no difference in hemodynamics, lung mechanics or arterial blood gases between hMSC treated animals and controls. Subsequent studies are required to determine if the observed decrease in inflammatory transcription factors will translate into improvement in inflammation and in physiological parameters over the long term.

Regulation Policy for Cell and Tissue Therapies in Australia.

In Australia, therapeutic goods are regulated by the Australian Government through the Therapeutic Goods Administration. In recent times, there have been significant changes to legislative instruments to accommodate the emergence of biotherapies, and a new regulatory framework was implemented in 2013. Therapeutic goods are regulated as medicines, devices, or biologics. With some exemptions and exceptions, human cellular and tissue products are regulated as biologics. The new regulation has taken a risk-based approach, with the level of regulation reflecting the perceived risk of the product, and products are classified from class 1 (low risk) to class 4 (highest risk). Other than for class 1 products, manufacturers must demonstrate conformance with the Australian Code of Good Manufacturing Practice and compliance with relevant Therapeutic Goods Orders and Standards through the submission of dossiers. The higher the classification of product, the more rigorous the dossier requirements. Listing of products on the Australian Therapeutic Goods Register is required for all products with some exceptions. The new regulatory framework provides greater clarity for manufacturers and strives to harmonize with international standards.

Adult human mesenchymal stromal cells and the treatment of graft versus host disease.

Graft versus host disease is a difficult and potentially lethal complication of hematopoietic stem cell transplantation. It occurs with minor human leucocyte antigen (HLA) mismatch and is normally treated with corticosteroid and other immunosuppressive therapy. When it is refractory to steroid therapy, mortality approaches 80%. Mesenchymal stromal cells are rare cells found in bone marrow and other tissues. They can be expanded in culture and possess complex and diverse immunomodulatory activity. Moreover, human mesenchymal stromal cells carry low levels of class 1 and no class 2 HLA antigens, making them immunoprivileged and able to be used without HLA matching. Their use in steroid-refractory graft versus host disease was first described in 2004. Subsequently, they have been used in a number of Phase I and II trials in acute and chronic graft versus host disease trials with success. We discuss their mode of action, the results, their production, and potential dangers with a view to future application.

Long live the stem cell: the use of stem cells isolated from post mortem tissues for translational strategies.

The "stem cell" has become arguably one of the most important biological tools in the arsenal of translational research directed at regeneration and repair. It remains to be seen whether every tissue has its own stem cell niche, although relatively recently a large amount of research has focused on isolating and characterizing tissue-specific stem cell populations, as well as those that are able to be directed to transdifferentiate into a variety of different lineages. Traditionally, stem cells are isolated from the viable tissue of embryonic, fetal, or adult living hosts; from "fresh" donated tissues that have been surgically or otherwise removed (biopsies), or obtained directly from tissues within minutes to several hours post mortem (PM). These human progenitor/stem cell sources remain potentially highly controversial, since they are accompanied by various still-unresolved ethical, social, moral and legal challenges. Due to the limited number of "live" donors, the small amount of material obtained from biopsies and difficulties during purification processes, harvesting from cadaveric material presents itself as an alternative strategy that could provide a hitherto untapped source of stem cells. However, PM stem cells are not without their own unique set of limitations including difficulty of obtaining samples, limited supply of material, variations in delay between death and sample collection, possible lack of medication history and suboptimal retrospective assignment of diagnostic and demographic data. This article is part of a Directed Issue entitled: Regenerative Medicine: The challenge of translation.

Mesenchymal stromal cell therapy for steroid-refractory acute and chronic graft versus host disease: a phase 1 study.

Steroid-refractory acute graft versus host disease (AGVHD) and chronic graft versus host disease (CGVHD) after allogeneic haematopoietic stem cell transplantation are major causes of morbidity and mortality. We undertook a phase I trial in patients with steroid-refractory AGVHD and CGVHD utilising bone marrow-derived mesenchymal stromal cells (MSC). Additionally, all refractory patients were treated with etanercept concomitantly. The primary end point was safety, and secondary end points were best response achieved and overall survival. A median of two infusions per patient were administered. The response rate overall for AGVHD was complete in seven, partial in four and no response in one patient. Of the seven patients who achieved a complete response, six are alive. The actuarial survival for the overall group of AGVHD was 55% at 30 months. Two patients with CGVHD achieved complete response with two partial responses and three with no response. The survival for those with AGVHD who achieved a complete response compared with those who did not was significant (p = 0.03). We identified no early or late safety issues in the nineteen patients. In view of the poor outlook for steroid-refractory AGVHD, further trials are warranted of MSC with steroid therapy, at the onset of AGVHD before steroid resistance.

Identification of three novel plasminogen (PLG) gene mutations in a series of 23 patients with low PLG activity.

Inherited severe hypoplasminogenaemia is a multisystemic disorder leading to deficient extravascular fibrinolysis. As a clinical consequence wound healing capacity of mucous membranes is markedly impaired leading to ligneous conjunctivitis and several other manifestations. Here we report the molecular genetic and clinical findings on 23 new cases with severe hypoplasminogenaemia. Homozygous or compound-heterozygous mutations in the plasminogen (PLG) gene were found in 16 of 23 patients (70%), three of which were novel mutations reported here for the first time (C166Y, Y264S, IVS10-7T/G). Compared to 79 previously published cases, clinical manifestations of the current group of patients showed higher percentages of ligneous periodontitis, congenital hydrocephalus, and involvement of the female genital tract. In contrast, involvement of the gastrointestinal or urogenital tract was not observed in any of the cases. Patients originated to a large extent (61%) from Turkey and the Middle East, and showed a comparably frequent occurrence of consanguinity of affected families and a greater female to male ratio than was derived from previous reports in the literature. Individual treatment of ligneous conjunctivitis included topical plasminogen or heparin eye drops, topical or systemic fresh frozen plasma, and surgical removal of ligneous pseudomembranes, mostly with modest or transient efficacy. In conclusion, the present study underscores the broad range of clinical manifestations in PLG-deficient patients with a trend to regional differences. Transmission of genetic and clinical data to the recently established Plasminogen Deficiency Registry should help to determine the prevalence of the disease and to develop more efficient treatment strategies.