Roberto S. Oliveri

Enrichment of autologous fat grafts with ex-vivo expanded adipose tissue-derived stem cells for graft survival: a randomised placebo-controlled trial

BACKGROUND Autologous fat grafting is increasingly used in reconstructive surgery. However, resorption rates ranging from 25% to 80% have been reported. Therefore, methods to increase graft viability are needed. Here, we report the results of a triple-blind, placebo-controlled trial to compare the survival of fat grafts enriched with autologous adipose-derived stem cells (ASCs) versus non-enriched fat grafts. METHODS Healthy participants underwent two liposuctions taken 14 days apart: one for ASC isolation and ex-vivo expansion, and another for the preparation of fat grafts. Two purified fat grafts (30 mL each) taken from the second liposuction were prepared for each participant. One graft was enriched with ASCs (20 × 10(6) cells per mL fat), and another graft without ASC enrichment served as a control. The fat grafts were injected subcutaneously as a bolus to the posterior part of the right and left upper arm according to the randomisation sequence. The volumes of injected fat grafts were measured by MRI immediately after injection and after 121 days before surgical removal. The primary goal was to compare the residual graft volumes of ASC-enriched grafts with those of control grafts. This study is registered at www.clinicaltrialsregister.eu, number 2010-023006-12. FINDINGS 13 participants were enrolled, three of whom were excluded. Compared with the control grafts, the ASC-enriched fat grafts had significantly higher residual volumes: 23·00 (95% CI 20·57-25·43) cm(3) versus 4·66 (3·16-6·16) cm(3) for the controls, corresponding to 80·9% (76·6-85·2) versus 16·3% (11·1-21·4) of the initial volumes, respectively (p<0·0001). The difference between the groups was 18·34 (95% CI 15·70-20·98) cm(3), equivalent to 64·6% (57·1-72·1; p<0·0001). No serious adverse events were noted. INTERPRETATION The procedure of ASC-enriched fat grafting had excellent feasibility and safety. These promising results add significantly to the prospect of stem cell use in clinical settings, and indicate that ASC graft enrichment could render lipofilling a reliable alternative to major tissue augmentation, such as breast surgery, with allogeneic material or major flap surgery. FUNDING Danish Cancer Society, Centre of Head and Orthopaedics Rigshospitalet, and Moalem Weitemeyer Bendtsen.

How I treat patients with massive hemorrhage.

Massive hemorrhage is associated with coagulopathy and high mortality. The transfusion guidelines up to 2006 recommended that resuscitation of massive hemorrhage should occur in successive steps using crystalloids, colloids, and red blood cells (RBCs) in the early phase and plasma and platelets in the late phase. With the introduction of the cell-based model of hemostasis in the mid-1990s, our understanding of the hemostatic process and of coagulopathy has improved. This has contributed to a change in resuscitation strategy and transfusion therapy of massive hemorrhage along with an acceptance of the adequacy of whole blood hemostatic tests to monitor these patients. Thus, in 2005, a strategy aiming at avoiding coagulopathy by proactive resuscitation with blood products in a balanced ratio of RBC:plasma:platelets was introduced, and this has been reported to be associated with reduced mortality in observational studies. Concurrently, whole blood viscoelastic hemostatic assays have gained acceptance by allowing a rapid and timely identification of coagulopathy along with enabling an individualized, goal-directed transfusion therapy. These strategies joined together seem beneficial for patient outcome, although final evidence on outcome from randomized controlled trials are lacking. We present how we in Copenhagen and Houston, today, manage patients with massive hemorrhage.

Mesenchymal stem cells improve locomotor recovery in traumatic spinal cord injury: systematic review with meta-analyses of rat models.

Traumatic spinal cord injury (SCI) is a devastating event with huge personal and societal costs. A limited number of treatments exist to ameliorate the progressive secondary damage that rapidly follows the primary mechanical impact. Mesenchymal stem or stromal cells (MSCs) have anti-inflammatory and neuroprotective effects and may thus reduce secondary damage after administration. We performed a systematic review with quantitative syntheses to assess the evidence of MSCs versus controls for locomotor recovery in rat models of traumatic SCI, and identified 83 eligible controlled studies comprising a total of 1,568 rats. Between-study heterogeneity was large. Fifty-three studies (64%) were reported as randomised, but only four reported adequate methodologies for randomisation. Forty-eight studies (58%) reported the use of a blinded outcome assessment. A random-effects meta-analysis yielded a difference in behavioural Basso-Beattie-Bresnahan (BBB) locomotor score means of 3.9 (95% confidence interval [CI] 3.2 to 4.7; P<0.001) in favour of MSCs. Trial sequential analysis confirmed the findings of the meta-analyses with the upper monitoring boundary for benefit being crossed by the cumulative Z-curve before reaching the diversity-adjusted required information size. Only time from intervention to last follow-up remained statistically significant after adjustment using multivariate random-effects meta-regression modelling. Lack of other demonstrable explanatory variables could be due to insufficient meta-analytic study power. MSCs would seem to demonstrate a substantial beneficial effect on locomotor recovery in a widely-used animal model of traumatic SCI. However, the animal results should be interpreted with caution concerning the internal and external validity of the studies in relation to the design of future clinical trials.

Pooled human platelet lysate versus fetal bovine serum—investigating the proliferation rate, chromosome stability and angiogenic potential of human adipose tissue-derived stem cells intended for clinical use

BACKGROUND AIMS Because of an increasing focus on the use of adipose-derived stem cells (ASCs) in clinical trials, the culture conditions for these cells are being optimized. We compared the proliferation rates and chromosomal stability of ASCs that had been cultured in Dulbeccos modified Eagles Medium (DMEM) supplemented with either pooled human platelet lysate (pHPL) or clinical-grade fetal bovine serum (FBS) (DMEM(pHPL) versus DMEM(FBS)). METHODS ASCs from four healthy donors were cultured in either DMEM(pHPL) or DMEM(FBS), and the population doubling time (PDT) was calculated. ASCs from two of the donors were expanded in DMEM(pHPL) or DMEM(FBS) and cultured for the final week before harvesting with or without the addition of vascular endothelial growth factor. We assessed the chromosomal stability (through the use of array comparative genomic hybridization), the expression of ASC and endothelial surface markers and the differentiation and angiogenic potential of these cells. RESULTS The ASCs that were cultured in pHPL exhibited a significantly shorter PDT of 29.6 h (95% confidence interval, 22.3-41.9 h) compared with those cultured in FBS, for which the PDT was 123.9 h (95% confidence interval, 95.6-176.2 h). Comparative genomic hybridization analyses revealed no chromosomal aberrations. Cell differentiation, capillary structure formation and cell-surface marker expression were generally unaffected by the type of medium supplement that was used or by the addition of vascular endothelial growth factor. CONCLUSIONS We observed that the use of pHPL as a growth supplement for ASCs facilitated a significantly higher proliferation rate compared with FBS without compromising genomic stability or differentiation capacity.

Hemostatic resuscitation with plasma and platelets in trauma

Background: Continued hemorrhage remains a major contributor of mortality in massively transfused patients and controversy regarding the optimal management exists although recently, the concept of hemostatic resuscitation, i.e., providing large amount of blood products to critically injured patients in an immediate and sustained manner as part of an early massive transfusion protocol has been introduced. The aim of the present review was to investigate the potential effect on survival of proactive administration of plasma and/or platelets (PLT) in trauma patients with massive bleeding. Materials and Methods: English databases were searched for reports of trauma patients receiving massive transfusion (10 or more red blood cell (RBC) within 24 hours or less from admission) that tested the effects of administration of plasma and/or PLT in relation to RBC concentrates on survival from January 2005 to November 2010. Comparison between highest vs lowest blood product ratios and 30-day mortality was performed. Results: Sixteen studies encompassing 3,663 patients receiving high vs low ratios were included. This meta-analysis of the pooled results revealed a substantial statistical heterogeneity (I2 = 58%) and that the highest ratio of plasma and/or PLT or to RBC was associated with a significantly decreased mortality (OR: 0.49; 95% confidence interval: 0.43-0.57; P<0.0001) when compared with lowest ratio. Conclusion: Meta-analysis of 16 retrospective studies concerning massively transfused trauma patients confirms a significantly lower mortality in patients treated with the highest fresh frozen plasma (FFP) and/or PLT ratio when compared with the lowest FFP and/or PLT ratio. However, optimal ranges of FFP: RBC and PLT : RBC should be established in randomized controlled trials.

Regional differences in expression of specific markers for human embryonic stem cells

Characterization of human embryonic stem cell (hESC) lines derived from the inner cell masses of blastocysts generally includes expression analysis of markers such as OCT4, NANOG, SSEA3, SSEA4, TRA-1-60 and TRA-1-81. Expression is usually detected by immunocytochemical staining of entire colonies of hESC, using one colony for each individual marker. Four newly established hESC lines showed the expected expression pattern and were capable of differentiating into the three germ layers in vitro. Neighbouring sections of entire colonies grown for 4, 11, 21 and 28 days respectively were stained with different markers to study the regional distribution and cellular co-expression. TRA-1-60 staining defined the hESC territory at all time points analysed. This territory comprised a characteristic OCT4 and NANOG staining often in overlapping subregions. Staining intensity of nuclei varied from strong OCT4 staining to weak or absent NANOG staining, and vice versa. SSEA4 staining was only observed in small clusters or single cells and not confined to the TRA territory. Co-expression of all markers was only detected in small areas. SSEA1 expression was found exclusively outside the TRA territory. In conclusion, pronounced regional differences in the expression of markers considered specific for undifferentiated hESC may suggest the existence of different cell populations.

Mesenchymal stem cell therapy for salivary gland dysfunction and xerostomia: a systematic review of preclinical studies.

The most severe forms of xerostomia and salivary gland dysfunction, as well as a severely reduced quality of life, are seen in Sjögren syndrome (SS) and after radiotherapy for head and neck cancer. For both conditions, no effective regenerative therapies yet exist. Thus, the aim of this article was to assess, through systematic review, the potential benefit of mesenchymal stem cell (MSC) therapy in radiation-induced and SS-related salivary gland dysfunction and xerostomia. We searched PubMed/MEDLINE, Embase, Web of Science, the Cochrane Database of Systematic Reviews, the World Health Organization Clinical Trials Registry Platform, and Google Scholar. We identified 6 separate study comparisons eligible for inclusion. Owing to the limited number of studies, we conclude that more randomized, adequately powered clinical trials are needed to validate the potential beneficial effect of MSCs on salivary gland dysfunction and xerostomia. Nonetheless, the preliminary studies identified in the present review were encouraging for further research.

Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions.

This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging onto 16-well glass chambers, and continuing with the general IF and qPCR steps will be provided. The techniques will be illustrated with new results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, and PDGF signaling pathways to primary cilia in stem cell maintenance and differentiation. Furthermore, a sample qPCR experiment will be shown illustrating that these techniques can be important tools in answering basic questions about hESC biology.

The use of viscoelastic haemostatic assays in goal-directing treatment with allogeneic blood products – A systematic review and meta-analysis

BackgroundManagement of the critically bleeding patient can be encountered in many medical and surgical settings. Common for these patients is a high risk of dying from exsanguination secondary to developing coagulopathy. The purpose of this meta-analysis was to systematically review and assess randomised controlled trials (RCTs) performed on patients in acute need for blood transfusions due to bleeding to evaluate the effect of viscoelastic haemostatic assay (VHA) guidance on bleeding, transfusion requirements and mortality.MethodsPubMed and EMBASE were searched for RCTs that 1) randomised patients into receiving transfusions based on either a VHA-guided (thromboelastography [TEG] or rotational thromboelastometry [ROTEM]) algorithm (intervention group) or at the clinician’s discretion and/or based on conventional coagulation tests (control group) and 2) adequately reported on the outcomes bleeding and/or transfusions and/or mortality. Data on bleeding, transfusions and mortality were extracted from each trial and included in a meta-analysis.ResultsFifteen RCTs (n = 1238 patients) were included. Nine trials referred to cardiothoracic patients, one to liver transplantation, one to surgical excision of burn wounds and one to trauma. One trial was conducted with cirrhotic patients, one with patients undergoing scoliosis surgery while one trial randomised treatment in post-partum females presenting with bleeding. The amount of transfused red blood cells (RBCs), fresh frozen plasma (FFP) and bleeding volume was found to be significantly reduced in the VHA-guided groups, whereas no significant difference was found for platelet transfusion requirements or mortality.

First-in-man mesenchymal stem cells for radiation-induced xerostomia (MESRIX): study protocol for a randomized controlled trial

BackgroundSalivary gland hypofunction and xerostomia are major complications following radiotherapy for head and neck cancer and may lead to debilitating oral disorders and impaired quality of life. Currently, only symptomatic treatment is available. However, mesenchymal stem cell (MSC) therapy has shown promising results in preclinical studies. Objectives are to assess safety and efficacy in a first-in-man trial on adipose-derived MSC therapy (ASC) for radiation-induced xerostomia.MethodsThis is a single-center, phase I/II, randomized, placebo-controlled, double-blinded clinical trial. A total of 30 patients are randomized in a 1:1 ratio to receive ultrasound-guided, administered ASC or placebo to the submandibular glands. The primary outcome is change in unstimulated whole salivary flow rate. The secondary outcomes are safety, efficacy, change in quality of life, qualitative and quantitative measurements of saliva, as well as submandibular gland size, vascularization, fibrosis, and secretory tissue evaluation based on contrast-induced magnetic resonance imaging (MRI) and core-needle samples. The assessments are performed at baseline (1 month prior to treatment) and 1 and 4 months following investigational intervention.DiscussionThe trial is the first attempt to evaluate the safety and efficacy of adipose-derived MSCs (ASCs) in patients with radiation-induced xerostomia. The results may provide evidence for the effectiveness of ASC in patients with salivary gland hypofunction and xerostomia and deliver valuable information for the design of subsequent trials.Trial registrationEudraCT, Identifier: 2014-004349-29. Registered on 1 April 2015.ClinicalTrials.gov, Identifier: NCT02513238. First received on 2 July 2015.The trial is prospectively registered.