In Vivo Confocal Microscopy in Limbal Stem Cell Deficiency After Mesenchymal Stem Cell Transplantation: A Sub-analysis from a Phase I-II Clinical Trial.

INTRODUCTION: The aim of this work is to evaluate the effect of mesenchymal stem cell transplantation (MSCT) and cultivated limbal epithelial transplantation (CLET) therapies on the limbus of patients suffering from limbal stem cell deficiency (LSCD). METHODS: A sub-analysis of a phase I-II randomized, controlled, and double-masked clinical trial was performed to assess the changes in the anatomical structures of the limbus. In vivo confocal microscopy (IVCM) analysis was carried out in LSCD eyes before and 12 months after allogeneic MSCT or CLET. Epithelial phenotype of the central cornea, as well as the presence of transition zones and palisades of Vogt in the limbus, were assessed using Wilcoxon test. RESULTS: Twenty-three LSCD (14 MSCT and nine CLET) eyes were included. The epithelial phenotype of the central cornea improved significantly (p < 0.001) from 15 (eight MSCT, seven CLET) and eight (six MSCT, two CLET) LSCD eyes showing conjunctival and mixed phenotypes, respectively, to eight (five MSCT, three CLET), five (two MSCT, three CLET), and ten (seven MSCT, three CLET) eyes showing conjunctival, mixed, and corneal phenotypes, respectively. Transition areas and palisades of Vogt were observed in at least one quadrant in nine (five MSCT, four CLET) and 16 (nine MSCT, seven CLET), and in four (two MSCT, two CLET) and six (three MSCT, three CLET) LSCD eyes before and after surgery, respectively. Changes in the transition zones and palisades were solely significant (p = 0.046) for the nasal and inferior quadrants, respectively. CONCLUSIONS: MSCT and CLET improved the central corneal epithelial phenotype despite only minor changes in the anatomical structures of the limbus, as detected by IVCM technology. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT01562002.

Long-term efficacy of autologous bone marrow mesenchymal stromal cells for treatment of knee osteoarthritis.

Knee osteoarthritis is the most prevalent joint disease and a frequent cause of pain, functional loss and disability. Conventional treatments have demonstrated only modest clinical benefits whereas cell-based therapies have shown encouraging results, but important details, such as dose needed, long-term evolution or number of applications required are scarcely known. Here we have reanalyzed results from two recent pilot trials with autologous bone marrow-derived mesenchymal stromal cells using the Huskisson plot to enhance quantification of efficacy and comparability. We find that cell doses of 10, 40 and 100 million autologous cells per knee provided quite similar healing results and that much of the effect attained 1 year after cell application remained after 2 and 4 years. These results are encouraging because they indicate that, apart from safety and simplicity: (i) the beneficial effect is both significant and sizeable, (ii) it can be achieved with a single injection of cells, and (iii) the effect is perdurable for years.Trial registration: EudraCT 2009-017405-11; NCT02123368. Registered 25 April 2014-Prospectively registered, https://clinicaltrials.gov/ct2/show/NCT02123368?term=02123368&draw=2&rank=1.

Bone regeneration with autologous adipose-derived mesenchymal stem cells: A reliable experimental model in rats.

The adult mesenchymal stem cell (MSC) has been proposed to be the definitive tool in regenerative medicine due to its multi-differentiation potential and expansion capacity ex vivo. The use of MSCs on bone regeneration has been assessed in several studies, obtaining promising results. However, the endless combinations that can be tested and the heterogeneity in the experimental conditions become a drawback when comparing results between authors. Moreover, it is very hard to find autologous studies using adipose-derived MSCs (AD-MSC) in rodents, which is the most used preclinical animal model. In this article an experimental model for basic bone tissue engineering research is described and justified, on which adult AD-MSCs are safely isolated from the rat dorsal interscapular fat pad, allowing ex vivo expansion and autogenous orthotopic reimplantation in a bilateral mandibular bone defect made in the same animal. This reliable and reproducible model provides a simple way to perform basic experimentation studies in a small animal model using autologous MSC for bone regeneration or cell therapy techniques prior to improve the research on large animal models.•Predictable and safe harvest of adipose-derived MSC. No need of animal sacrifice.•Allows for autologous studies with the most frequently used animal model: the rat. No need of allogeneic or human MSC use and, therefore, immunological concerns are avoided.•Bilateral mandibular critical size defect to allow direct control/experimental comparison.

Autologous bone marrow expanded mesenchymal stem cells in patellar tendinopathy: protocol for a phase I/II, single-centre, randomized with active control PRP, double-blinded clinical trial.

INTRODUCTION: Patellar tendon overuse injuries are common in athletes. Imaging may show a change in tissue structure with tendon thickening and disruption of the intratendinous substance. We wish to test the hypothesis that both autologous bone marrow expanded mesenchymal stem cells and autologous leukocyte-poor platelet-rich plasma (LP-PRP) implanted into the area of the disrupted tendinopathic patellar tendon will restore function, but tendon regeneration tissue will only be observed in the subjects treated with autologous bone marrow expanded mesenchymal stem cells. METHODS AND ANALYSIS: This is a single-centre, pilot phase I/II, double-blinded clinical trial with randomisation with active control. Twenty patients with a diagnosis of patellar tendinopathy with imaging changes (tendon thickening and disruption of the intratendinous substance at the proximal portion of the patellar tendon) will be randomised in a 1:1 ratio to receive a local injection of either bone-marrow autologous mesenchymal stem cells (MSC), isolated and cultured under GMP at The Institute of Biology and Molecular Genetics (IBGM) (Spain) or P-PRP. The study will have two aims: first, to ascertain whether a clinically relevant improvement after 3, 6 and 12 months according to the visual analogue scale (VAS), Victorian Institute of Sport Assessment for patellar tendons (VISA-P) and dynamometry scales (DYN) will be achieved; and second, to ascertain whether the proposed intervention will restore tendon structure as determined by ultrasonography (US), Doppler ultrasonography (DUS), and innovative MRI and ultrasound techniques: Magnetic Resonance T2 FAT SAT (UTE, Ultrashort Echo TE) sequence and Ultrasound Tissue Characterization (UTC). Patients who are randomised to the P-PRP treatment group but do not achieve a satisfactory primary endpoint after 6 months will be offered treatment with MSC. TRIAL REGISTRATION: NCT03454737.

A proof-of-concept clinical trial using mesenchymal stem cells for the treatment of corneal epithelial stem cell deficiency.

Ocular stem cell transplantation derived from either autologous or allogeneic donor corneoscleral junction is a functional cell therapy to manage extensive and/or severe limbal stem cell deficiencies that lead to corneal epithelial failure. Mesenchymal stem cells have been properly tested in animal models of this ophthalmic pathology, but never in human eyes despite their potential advantages. We conducted a 6- to 12-month proof-of-concept, randomized, and double-masked pilot trial to test whether allogeneic bone marrow-derived mesenchymal stem cell transplantation (MSCT], n = 17) was as safe and as equally efficient as allogeneic cultivated limbal epithelial transplantation (CLET), (n = 11) to improve corneal epithelial damage due to limbal stem cell deficiency. Primary endpoints demanded combination of symptoms, signs, and the objective improvement of the epithelial phenotype in central cornea by in vivo confocal microscopy. This proof-of-concept trial showed that MSCT was as safe and efficacious as CLET. Global success at 6-12 months was 72.7%-77.8% for CLET cases and 76.5%-85.7% for MSCT cases (not significant differences). Central corneal epithelial phenotype improved in 71.4% and 66.7% of MSCT and CLET cases, respectively at 12 months (P = 1.000). There were no adverse events related to cell products. This trial suggests first evidence that MSCT facilitated improvement of a diseased corneal epithelium due to lack of its stem cells as efficiently as CLET. Consequently, not only CLET but also MSCT deserves more preclinical investigational resources before the favorable results of this proof-of-concept trial could be transformed into the larger numbers of the multicenter trials that would provide stronger evidence. (ClinicalTrials.gov number, NCT01562002.).

Intervertebral Disc Repair by Allogeneic Mesenchymal Bone Marrow Cells: A Randomized Controlled Trial.

BACKGROUND: Degenerative disc disease often causes severe low-back pain, a public health problem with huge economic and life quality impact. Chronic cases often require surgery, which may lead to biomechanical problems and accelerated degeneration of the adjacent segments. Autologous mesenchymal stromal cells (MSC) treatments have shown feasibility, safety and strong indications of clinical efficacy. We present here a randomized, controlled trial using allogeneic MSC, which are logistically more convenient than autologous cells. METHODS: We randomized 24 patients with chronic back pain diagnosed with lumbar disk degeneration and unresponsive to conservative treatments into 2 groups. The test group received allogeneic bone marrow MSCs by intradiscal injection of 25 × 10 cells per segment under local anesthesia. The control group received a sham infiltration of paravertebral musculature with the anesthetic. Clinical outcomes were followed up for 1 year and included evaluation of pain, disability, and quality of life. Disc quality was followed up by magnetic resonance imaging. RESULTS: Feasibility and safety were confirmed and indications of clinical efficacy were identified. MSC-treated patients displayed a quick and significant improvement in algofunctional indices versus the controls. This improvement seemed restricted to a group of responders that included 40% of the cohort. Degeneration, quantified by Pfirrmann grading, improved in the MSC-treated patients and worsened in the controls. CONCLUSIONS: Allogeneic MSC therapy may be a valid alternative for the treatment of degenerative disc disease that is more logistically convenient than the autologous MSC treatment. The intervention is simple, does not require surgery, provides pain relief, and significantly improves disc quality.

Treatment of Knee Osteoarthritis With Allogeneic Bone Marrow Mesenchymal Stem Cells: A Randomized Controlled Trial.

BACKGROUND: Osteoarthritis is the most prevalent joint disease and a common cause of joint pain, functional loss, and disability. Conventional treatments demonstrate only modest clinical benefits without lesion reversal. Autologous mesenchymal stromal cell (MSC) treatments have shown feasibility, safety, and strong indications for clinical efficacy. We performed a randomized, active control trial to assess the feasibility and safety of treating osteoarthritis with allogeneic MSCs, and we obtain information regarding the efficacy of this treatment. METHODS: We randomized 30 patients with chronic knee pain unresponsive to conservative treatments and showing radiological evidence of osteoarthritis into 2 groups of 15 patients. The test group was treated with allogeneic bone marrow MSCs by intra-articular injection of 40 × 10(6) cells. The control group received intra-articular hyaluronic acid (60 mg, single dose). Clinical outcomes were followed for 1 year and included evaluations of pain, disability, and quality of life. Articular cartilage quality was assessed by quantitative magnetic resonance imaging T2 mapping. RESULTS: Feasibility and safety were confirmed and indications of clinical efficacy were identified. The MSC-treated patients displayed significant improvement in algofunctional indices versus the active controls treated with hyaluronic acid. Quantification of cartilage quality by T2 relaxation measurements showed a significant decrease in poor cartilage areas, with cartilage quality improvements in MSC-treated patients. CONCLUSIONS: Allogeneic MSC therapy may be a valid alternative for the treatment of chronic knee osteoarthritis that is more logistically convenient than autologous MSC treatment. The intervention is simple, does not require surgery, provides pain relief, and significantly improves cartilage quality.

Treatment of knee osteoarthritis with autologous mesenchymal stem cells: a pilot study.

BACKGROUND: Osteoarthritis is the most prevalent joint disease and a frequent cause of joint pain, functional loss, and disability. Osteoarthritis often becomes chronic, and conventional treatments have demonstrated only modest clinical benefits without lesion reversal. Cell-based therapies have shown encouraging results in both animal studies and a few human case reports. We designed a pilot study to assess the feasibility and safety of osteoarthritis treatment with mesenchymal stromal cells (MSCs) in humans and to obtain early efficacy information for this treatment. METHODS: Twelve patients with chronic knee pain unresponsive to conservative treatments and radiologic evidence of osteoarthritis were treated with autologous expanded bone marrow MSCs by intra-articular injection (40×10 cells). Clinical outcomes were followed for 1 year and included evaluations of pain, disability, and quality of life. Articular cartilage quality was assessed by quantitative magnetic resonance imaging T2 mapping. RESULTS: Feasibility and safety were confirmed, and strong indications of clinical efficacy were identified. Patients exhibited rapid and progressive improvement of algofunctional indices that approached 65% to 78% by 1 year. This outcome compares favorably with the results of conventional treatments. Additionally, quantification of cartilage quality by T2 relaxation measurements demonstrated a highly significant decrease of poor cartilage areas (on average, 27%), with improvement of cartilage quality in 11 of the 12 patients. CONCLUSIONS: MSC therapy may be a valid alternative treatment for chronic knee osteoarthritis. The intervention is simple, does not require hospitalization or surgery, provides pain relief, and significantly improves cartilage quality.

Intervertebral disc repair by autologous mesenchymal bone marrow cells: a pilot study.

BACKGROUND: Degenerative disc disease may cause severe low-back pain, a large public health problem with significant economic and life quality impact. Chronic cases often require surgery, which may lead to biomechanical problems and accelerated degeneration of the adjacent segments. Cell-based therapies may circumvent these problems and have exhibited encouraging results in vitro and in animal studies. We designed a pilot study to assess feasibility and safety and to obtain early indications on efficacy of treatment with mesenchymal stem cells (MSC) in humans. METHODS: Ten patients with chronic back pain diagnosed with lumbar disc degeneration with intact annulus fibrosus were treated with autologous expanded bone marrow MSC injected into the nucleus pulposus area. Clinical evolution was followed for 1 year and included evaluation of back pain, disability, and quality of life. Magnetic resonance imaging measurements of disc height and fluid content were also performed. RESULTS: Feasibility and safety were confirmed and strong indications of clinical efficacy identified. Patients exhibited rapid improvement of pain and disability (85% of maximum in 3 months) that approached 71% of optimal efficacy. This outcome compares favorably with the results of other procedures such as spinal fusion or total disc replacement. Although disc height was not recovered, water content was significantly elevated at 12 months. CONCLUSIONS: MSC therapy may be a valid alternative treatment for chronic back pain caused by degenerative disc disease. Advantages over current gold standards include simpler and more conservative intervention without surgery, preservation of normal biomechanics, and same or better pain relief.

Prospective comparative analysis of the angiogenic capacity of monocytes and CD133+ cells in a murine model of hind limb ischemia.

BACKGROUND AIMS: The aim of this study was to compare prospectively the vasculogenic capacity of two cell sources, monocytes and CD133+ cells. METHODS: Cells were obtained from healthy donors by adherence or magnetic selection. Animals studies were performed in a model of hind limb ischemia and different groups were established according to type and number of cells infused. Revascularization was measured by sequential blood flow analysis using a laser Doppler device and by assessing capillary density in the ischemic muscles. In order to locate the infused cells, immunofluorescence and immunocytochemistry techniques were performed and analyzed by light and confocal microscopy. RESULTS: During the study period there was a significant improvement in both limb perfusion and capillary density in mice treated with either human monocytes or CD133+ cells (P<0.05) compared with non-treated mice. No cells were detected as incorporated into the vessels when 1 x 10(5) cells were used but with higher doses (1 x 10(6)) a few human cells were observed integrated into the vessels in both groups of treated mice. Supernatants of both cell types showed vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and platelet-derived growth factor- AB (PDGF-AB) expression. CONCLUSIONS: Treatment with human monocytes or CD133+ cells improves blood perfusion and capillary density in a murine model and both cell types seem to stimulate vasculogenesis in a fairly similar way.

Bortezomib induces selective depletion of alloreactive T lymphocytes and decreases the production of Th1 cytokines.

We explored the ability of the proteasome inhibitor bortezomib, which prevents nuclear factor kappaB (NF-kappaB) activation, to block T-cell activation, proliferation, and survival within alloreactive compared with resting T cells. For this purpose, T cells were stimulated with PHA, alphaCD3/alphaCD28, or allogeneic dendritic cells or through mixed lymphocyte cultures. NF-kappaB expression increased in activated T lymphocytes compared with resting T cells. Of interest, the higher the NF-kappaB expression, the more intense the proliferative blockade induced by bortezomib. Moreover, after mixed lymphocyte reaction (MLR) cultures, alloreactive T cells were 2 logs more sensitive to bortezomib-induced apoptosis than the resting T-cell counterpart. This effect was due to a selective induction of apoptosis among activated T cells that was related to caspase activation and cleavage of the antiapoptotic bcl-2 protein and was partially abolished by the addition of the pancaspase inhibitor Z-VAD-FMK. In addition, after secondary MLR, the number of activated T cells was significantly reduced among T lymphocytes previously cultured with bortezomib when cells from the same donor were used as stimulating cells. By contrast, when third-party donor cells were used as stimulating cells, no significant differences were observed between T lymphocytes previously exposed or not to the drug, indicating a highly specific depletion of T lymphocytes alloreactive against primary donor antigens. The addition of bortezomib decreased not only the proliferation and viability of activated T lymphocytes but also the levels of IFNgamma and IL-2, which were significantly decreased among activated T cells cultured with bortezomib at doses ranging from 10 to 100 nM. In conclusion, at concentrations reached in the clinical setting, bortezomib induces selective apoptosis and decreases Th1 response among alloreactive T lymphocytes while it barely affects unstimulated T cells. These results establish the basis for the clinical use of bortezomib in the management of graft-versus-host disease (GVHD).

Posttransplant hematopoiesis in patients undergoing sibling allogeneic stem cell transplantation reflects that of their respective donors although with a lower functional capability.

We tested the principle of whether patient long-term hematopoiesis following allogeneic stem cell transplantation (allo-SCT) reflects the characteristics of the hematopoiesis of their respective donor. For this purpose, we analyzed bone marrow (BM) hematopoiesis using long-term cultures (LTC), delta assays, and clonogeneic assays as well as CD34+ cells and their subsets by flow cytometry in a series of 37 patients undergoing allo-SCT, and we compared it to that of their respective human leukocyte antigen-matched sibling donors in a paired study performed more than 1 year after the transplant procedure. Interestingly, the main factor that influenced post-allo-SCT BM hematopoiesis in the long term was donor hematopoiesis. Nevertheless, compared to their respective donors, patients exhibited a significantly lower number of colony-forming units granulomonocytic, burst-forming units erythroid, and immature progenitors (CD34++/CD38dim/CD90+/CD133+ cells, LTC-initiating cells, and colonies generated in the delta assay). Moreover, BM stromal function was diminished in patients undergoing allo-SCT compared to their donors. In addition, the presence of chronic graft-versus-host disease under immunosuppressive treatment also conditioned an impaired hematopoietic function. In summary, our study shows that BM hematopoiesis evaluated more than 1 year after an allo-SCT mainly reproduces that of their respective donors, although with a significantly decreased in vitro activity.

Long-term bone marrow culture data are the most powerful predictor of peripheral blood progenitor cell mobilization in healthy donors.

BACKGROUND AND OBJECTIVES: There is wide interindividual variation in progenitor cell mobilization. The present study was aimed to analyze steady state hematopoiesis in healthy donors and its influence on hematopoietic progenitor cell (HPC) mobilization. DESIGN AND METHODS: Bone marrow (BM) was aspirated from 72 healthy donors prior to administration of recombinant human granulocyte colony-stimulating factor (G-CSF). Analyses of CD34+ cells and semisolid cultures as well as long-term cultures were performed from BM or leukapheresis products. RESULTS: Male donors showed a higher number of BFU-E (p=0.007) and committed progenitors (p=0.05), a better stromal layer (p=0.02), and higher long-term bone marrow culture (LT-BMC) counts (p<0.05) when compared to those in female donors. When correlating the culture pattern of the BM with the data from the leukapheresis products, we observed that the number of the immature progenitors in BM correlated significantly with both the number of CD34 + cells and CFU-GM in the first leukapheresis. Univariate analysis revealed that the following variables had a beneficial impact on the number of CD34+ cells: male sex, body weight >73 Kg, G-CSF schedule and results of LT-BMC, although in the multivariate analysis only the number of CFU-GM obtained after LT-BMC showed a significant influence (p<0.001). INTERPRETATION AND CONCLUSIONS: These results confirm the interindividual variation in HPC mobilization among healthy subjects, with LT-BMC counts being the most reliable predictor, expressing the behavior of the immature progenitors and their relationship with the microenvironment.

Mesenchymal stem cells are present in peripheral blood and can engraft after allogeneic hematopoietic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Whether human mesenchymal stem cells (MSC) can be transplanted is controversial and their presence in peripheral blood is not fully accepted. In the present study we have analyzed whether, within the allogeneic transplantation setting, MSC are of host or donor origin. DESIGN AND METHODS: Bone marrow MSC from 19 patients who had undergone allogeneic transplantation were expanded and identified using immunophenotypic markers. After that, chimerism studies were performed using reverse transcription polymerase chain reaction of short tandem repeat (STR) loci. Analyses were carried out at different time-points after transplantation, with a total of 44 samples studied. Bone marrow was used as the source of stem cells for transplantation in 4 cases and peripheral blood in 15 cases. The conditioning regimen was standard in 9 patients and non-myeloablative in 10 patients. RESULTS: Our results show that in the great majority of cases analyzed (17 out 19), MSC were of host origin. However, in 2 patients with multiple myeloma who had received a reduced intensity transplantation using peripheral blood stem cells, MSC were partially of donor origin (60.17% and 26.13% of total MSC). INTERPRETATION AND CONCLUSIONS: These findings indicate that after allogeneic transplantation MSC from the donor can engraft in bone marrow. Moreover, since the stem cells were obtained from peripheral blood, it can be concluded that MSC circulate among mobilized peripheral blood stem cells and can engraft in bone marrow after allogeneic transplantation.