Incremental benefits of repeated mesenchymal stromal cell administration compared with solitary intervention after myocardial infarction

Background aimsTraditionally, stem cell therapy for myocardial infarction (MI) has been administered as a single treatment in the acute or subacute period after MI. These time intervals coincide with marked differences in the post-infarct myocardial environment, raising the prospect that repeat cell dosing could provide incremental benefit beyond a solitary intervention. This prospect was evaluated with the use of mesenchymal stromal cells (MSCs).MethodsThree groups of rats were studied. Single-therapy and dual-therapy groups received allogeneic, prospectively isolated MSCs (1 × 10⁶ cells) by trans-epicardial injection immediately after MI, with additional dosing 1 week later in the dual-therapy cohort. Control animals received cryopreservant solution only. Left ventricular (LV) dimensions and ejection fraction (EF) were assessed by cardiac magnetic resonance immediately before MI and at 1, 2 and 4 weeks after MI.ResultsImmediate MSC treatment attenuated early myocardial damage with EF of 35.3 ± 3.1% (dual group, n = 12) and 35.2 ± 2.2% (single group, n = 15) at 1 week after MI compared with 22.1 ± 1.9% in controls (n = 17, P < 0.01). In animals receiving a second dose of MSCs, EF increased to 40.7 ± 3.1% by week 4, which was significantly higher than in the single-therapy group (EF 35.9 ± 1.8%, P < 0.05). Dual MSC treatment was also associated with greater myocardial mass and arteriolar density, with trends toward reduced myocardial fibrosis. These incremental benefits were especially observed in remote (non-infarct) segments of LV myocardium.ConclusionsRepeated stem cell intervention in both the acute and the sub-acute period after MI provides additional improvement in ventricular function beyond solitary cell dosing, largely owing to beneficial changes remote to the area of infarction.     

Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease

Background aimsGraft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects.MethodsThe ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rγ(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed.ResultsInjection of 200 × 10⁶ human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM₁ antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 10⁶ MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 × 10⁶ human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 10⁶ MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs.ConclusionsInjection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models.     

Identification and characterization of a large source of primary mesenchymal stem cells tightly adhered to bone surfaces of human vertebral body marrow cavities

BackgroundTherapeutic allogeneic mesenchymal stromal cells (MSCs) are currently in clinical trials to evaluate their effectiveness in treating many different disease indications. Eventual commercialization for broad distribution will require further improvements in manufacturing processes to economically manufacture MSCs at scales sufficient to satisfy projected demands. A key contributor to the present high cost of goods sold for MSC manufacturing is the need to create master cell banks from multiple donors, which leads to variability in large-scale manufacturing runs. Therefore, the availability of large single donor depots of primary MSCs would greatly benefit the cell therapy market by reducing costs associated with manufacturing.MethodsWe have discovered that an abundant population of cells possessing all the hallmarks of MSCs is tightly associated with the vertebral body (VB) bone matrix and only liberated by proteolytic digestion. Here we demonstrate that these vertebral bone-adherent (vBA) MSCs possess all the International Society of Cell and Gene Therapy-defined characteristics (e.g., plastic adherence, surface marker expression and trilineage differentiation) of MSCs, and we have therefore termed them vBA-MSCs to distinguish this population from loosely associated MSCs recovered through aspiration or rinsing of the bone marrow compartment.ResultsPilot banking and expansion were performed with vBA-MSCs obtained from 3 deceased donors, and it was demonstrated that bank sizes averaging 2.9 × 10⁸ ± 1.35 × 10⁸ vBA-MSCs at passage 1 were obtainable from only 5 g of digested VB bone fragments. Each bank of cells demonstrated robust proliferation through a total of 9 passages, without significant reduction in population doubling times. The theoretical total cell yield from the entire amount of bone fragments (approximately 300 g) from each donor with limited expansion through 4 passages is 100 trillion (1 × 10¹⁴) vBA-MSCs, equating to over 10⁵ doses at 10 × 10⁶ cells/kg for an average 70-kg recipient.DiscussionThus, we have established a novel and plentiful source of MSCs that will benefit the cell therapy market by overcoming manufacturing and regulatory inefficiencies due to donor-to-donor variability.     

Autologous bone marrow mesenchymal stromal cell therapy for “no-option” critical limb ischemia is limited by karyotype abnormalities

BackgroundCritical limb ischemia (CLI) is the most severe manifestation of peripheral vascular disease. Revascularization is the preferred therapy, but it is not achievable in 25%–40% of patients due to diffuse anatomic distribution of the disease or medical comorbidities. No-option CLI represents an unmet medical need. Mesenchymal stromal cells (MSCs) may provide salvage therapy through their angiogenic and tissue-trophic properties. This article reports a phase 1b clinical study examining the safety and feasibility of intramuscular transplantation of autologous bone-marrow MSCs for patients with no-option CLI.MethodsTwelve patients were enrolled in the clinical trial, and nine proceeded to bone marrow aspiration and culture expansion of MSCs.ResultsA high rate of karyotype abnormality (>30%) was detected in the produced cell batches, resulting in failure of release for clinical administration. Four patients were treated with the investigational medicinal product (IMP), three with a low dose of 20 × 10⁶ MSCs and one with a mid-dose of 40 × 10⁶ MSCs. There were no serious adverse events related to trial interventions, including bone marrow aspiration, IMP injection or therapy.ConclusionsThe results of this trial conclude that an autologous cell therapy approach with MSCs for critical limb ischemia is limited by the high rate of karyotype abnormalities.     

Pluripotent stem cell-derived mesenchymal stromal cells improve cardiac function and vascularity after myocardial infarction

Background aimsMesenchymal stromal cells (MSCs) have been shown to improve cardiac function after injury and are the subject of ongoing clinical trials. In this study, the authors tested the cardiac regenerative potential of an induced pluripotent stem cell-derived MSC (iPSC-MSC) population (Cymerus MSCs) in a rat model of myocardial ischemia-reperfusion (I/R). Furthermore, the authors compared this efficacy with bone marrow-derived MSCs (BM-MSCs), which are the predominant cell type in clinical trials.MethodsFour days after myocardial I/R injury, rats were randomly assigned to (i) a Cymerus MSC group (n = 15), (ii) a BM-MSC group (n = 15) or (iii) a vehicle control group (n = 14). For cell-treated animals, a total of 5 × 10⁶ cells were injected at three sites within the infarcted left ventricular (LV) wall.ResultsOne month after cell transplantation, Cymerus MSCs improved LV function (assessed by echocardiography) compared with vehicle and BM-MSCs. Interestingly, Cymerus MSCs enhanced angiogenesis without sustained engraftment or significant impact on infarct scar size. Suggesting safety, Cymerus MSCs had no effect on inducible tachycardia or the ventricular scar heterogeneity that provides a substrate for cardiac re-entrant circuits.ConclusionsThe authors here demonstrate that intra-myocardial administration of iPSC-MSCs (Cymerus MSCs) provide better therapeutic effects compared with conventional BM-MSCs in a rodent model of myocardial I/R. Because of its manufacturing scalability, iPSC-MSC therapy offers an exciting opportunity for an “off-the-shelf” stem cell therapy for cardiac repair.     

Efficacy and safety of mesenchymal stromal cell treatment from related donors for patients with refractory aplastic anemia

Background aimsThis study evaluated the feasibility, safety and immunological effects of the intravenous administration of mesenchymal stromal cells (MSCs) from a related donor in patients with refractory aplastic anemia (AA).MethodsA mean of 6 × 10⁵/kg (range, 5.0–7.1 × 10⁵) MSCs were injected intravenously to 18 patients, including 14 patients with nonsevere AA and four patients with severe AA who were refractory to prior immunosuppressive treatment. The outcomes of patients treated with MSCs were evaluated and compared with a historic control cohort, including 18 patients with refractory AA.ResultsTwo patients had injection-related adverse events, including transient fever and headache. No major adverse events were reported during the follow-up period. An immunological analysis revealed an increased proportion of CD4⁺CD25⁺ FOXP3⁺regulatory T cells in peripheral mononuclear cells. Following up for 1 year, six of 18 patients (33.3%) achieved a complete response or a partial response to MSC treatment. In six patients, two achieved a complete response including a recovery of three hematopoietic cell lines after MSCs therapy at days 88 and 92, two patients achieved only a red cell recovery with hemoglobin levels >100 g/L at days 30 and 48 and two patients had only a platelet recovery with a platelet count of >60 × 10⁹/L at days 54 and 81. In the control cohort, only one patient (5.56%) achieved a partial response during the follow-up period.ConclusionsThe data from the present study suggest that treatment with MSCs from a related donor may be a promising therapeutic strategy for patients with refractory AA. The trial has been registered at ClinicalTrials.gov: identifier NCT01305694.     

Ex vivo-expanded highly pure ABCB5+ mesenchymal stromal cells as Good Manufacturing Practice-compliant autologous advanced therapy medicinal product for clinical use: process validation and first in-human data

Background aimMesenchymal stromal cells (MSCs) hold promise for the treatment of tissue damage and injury. However, MSCs comprise multiple subpopulations with diverse properties, which could explain inconsistent therapeutic outcomes seen among therapeutic attempts. Recently, the adenosine triphosphate-binding cassette transporter ABCB5 has been shown to identify a novel dermal immunomodulatory MSC subpopulation.MethodsThe authors have established a validated Good Manufacturing Practice (GMP)-compliant expansion and manufacturing process by which ABCB5⁺ MSCs can be isolated from skin tissue and processed to generate a highly functional homogeneous cell population manufactured as an advanced therapy medicinal product (ATMP). This product has been approved by the German competent regulatory authority to be tested in a clinical trial to treat therapy-resistant chronic venous ulcers.ResultsAs of now, 12 wounds in nine patients have been treated with 5 × 10⁵ autologous ABCB5⁺ MSCs per cm² wound area, eliciting a median wound size reduction of 63% (range, 32–100%) at 12 weeks and early relief of pain.ConclusionsThe authors describe here their GMP- and European Pharmacopoeia-compliant production and quality control process, report on a pre-clinical dose selection study and present the first in-human results. Together, these data substantiate the idea that ABCB5⁺ MSCs manufactured as ATMPs could deliver a clinically relevant wound closure strategy for patients with chronic therapy-resistant wounds.     

In vivo evaluation of two types of bioactive scaffold used for tendon-bone interface healing in the reconstruction of anterior cruciate ligament

Fibrin glue combined with bone morphogenetic protein (BMP) and recombined bone xenograft (RBX), were compared to evaluate their effect on the tendon–bone interface healing. The interface of fibrin glue-BMP developed new cartilage but the new bone was thinner whereas the interface of RBX had large areas of chondrocyte-like cells, bone formation and an immature neo-enthesis structure. At 12 weeks, bone mineral density of RBX group (152 ± 52 cm³) and fibrin glue-BMP group (109 ± 13 cm³) was calculated by micro-computed tomography. The ultimate load of fibrin glue-BMP group (60 ± 18 and 51 ± 14 N) and RBX group (65 ± 21 and 57 ± 15 N) was shown by biomechanics at 6 and 12 weeks. RBX thus has an advantage on accelerating tendon–bone interface healing.     

Therapeutic effect of autologous bone marrow stem cell mobilization combined with anti-infective therapy on moyamoya disease

ObjectiveThe purpose of this study is to explore the therapeutic effect of autologous bone marrow stem cell (ABMSC) mobilization combined anti-infection therapy on patients with moyamoya disease (MMD), and to provide reference for the clinical treatment of MMD and cerebrovascular disease.Methods54 adult patients with MMD diagnosed in Henan Provincial People’s Hospital from March 2017 to March 2019 were chosen as research objects. All patients were randomly divided into study group (SG) and control group (CG), with 27 patients in each group. Patients in both groups received conventional drug treatment after diagnosis of MMD, and received dura turnover of brain - temporal muscle - superficial temporal artery application surgery during indirect vascular reconstruction. On the basis of surgical treatment, patients in the SG were given ABMSC mobilization combined with low-dose dexamethasone for anti-inflammatory and anti-infection treatment. ABMSCs were mobilized by recombinant human granulocyte colony stimulating factor (rhG-csF) and recombinant human granulocyte - macrophage colony stimulating factor (rhoM-esF). The therapeutic effects of the patients were evaluated BF, one month after treatment (AF), three months AF, and six months AF. The number of hematopoietic stem cells (HpCs) and inflammatory indicators were compared between the two groups before and 4 weeks AF.ResultsFirstly, the Barthcl index of patients in the two groups showed a gradual increase trend at the 3rd and 6th months AF, and the ascensional range in the research group was higher than that in the CG (P < 0.05). Secondly, at the 3rd and 6th month AF, national institute of heath stroke scale (NIHSS) scores of patients in the CG were lower than those before treatment (BF), and there was an important change in NIHSS scores between the two groups at the same period (P < 0.05). Thirdly, after 1 month of treatment and 3 months of treatment, Chinese stroke scale (CSS) scores of patients in both groups decreased obviously compared with those BF, and the SG was lower than the CG, with statistical changes (P < 0.05). Fourthly, after 4 weeks of treatment, the hematopoietic stem cell counts in both groups were higher than those BF, and the hematopoietic stem cell counts in the SG were obviously higher than those in the CG (P < 0.05). All three inflammatory indicators were improved compared with those BF, and the SG was better than the CG (P < 0.05).ConclusionAutogenous bone marrow stem cell mobilization combined with dexamethasone anti-inflammation and anti-infection treatment after revascularization in patients with MMD can accelerate the recovery of nerve function and promote the formation of new blood vessels. At the same time, it can reduce inflammation and improve patients' quality of life, which is worthy of clinical reference.     

Reliable isolation of human mesenchymal stromal cells from bone marrow biopsy specimens in patients after allogeneic hematopoietic cell transplantation

Isolation of mesenchymal stromal cells (MSCs) from pretreated, hematologic patients is challenging. Especially after allogeneic hematopoietic cell transplantation (HCT), standard protocols using bone marrow aspirates fail to reliably recover sufficient cell numbers. Because MSCs are considered to contribute to processes that mainly affect the outcome after transplantation, such as an efficient lymphohematopoietic recovery, extent of graft-versus-host disease as well as the occurrence of leukemic relapse, it is of great clinical relevance to investigate MSC function in this context. Previous studies showed that MSCs can be isolated by collagenase digestion of large bone fragments of hematologically healthy patients undergoing hip replacement or knee surgeries. We have now further developed this procedure for the isolation of MSCs from hematologic patients after allogeneic HCT by using trephine biopsy specimens obtained during routine examinations. Comparison of aspirates and trephine biopsy specimens from patients after allogeneic HCT revealed a significantly higher frequency of clonogenic MSCs (colony-forming unit–fibroblast [CFU-F]) in trephine biopsy specimens (mean, 289.8 ± standard deviation 322.5 CFU-F colonies/1 × 10⁶ total nucleated cells versus 4.2 ± 9.9; P < 0.0001). Subsequent expansion of functional MSCs isolated from trephine biopsy specimen was more robust and led to a significantly higher yield compared with control samples expanded from aspirates (median, 1.6 × 10⁶; range, 0–2.3 × 10⁷ P0 MSCs versus 5.4 × 10⁴; range, 0–8.9 × 10⁶; P < 0.0001). Using trephine biopsy specimens as MSC source facilitates the investigation of various clinical questions.     

ABCB5+ mesenchymal stromal cells facilitate complete and durable wound closure in recessive dystrophic epidermolysis bullosa

Background and aimsRecessive dystrophic epidermolysis bullosa (RDEB) is a hereditary, rare, devastating and life-threatening skin fragility disorder with a high unmet medical need. In a recent international, single-arm clinical trial, treatment of 16 patients (aged 6–36 years) with three intravenous infusions of 2 × 10⁶ immunomodulatory ABCB5⁺ dermal mesenchymal stromal cells (MSCs)/kg on days 0, 17 and 35 reduced disease activity, itch and pain. A post-hoc analysis was undertaken to assess the potential effects of treatment with ABCB5⁺ MSCs on the overall skin wound healing in patients suffering from RDEB.MethodsDocumentary photographs of the affected body regions taken on days 0, 17, 35 and at 12 weeks were evaluated regarding proportion, temporal course and durability of wound closure as well as development of new wounds.ResultsOf 168 baseline wounds in 14 patients, 109 (64.9%) wounds had closed at week 12, of which 63.3% (69 wounds) had closed already by day 35 or day 17. Conversely, 74.2% of the baseline wounds that had closed by day 17 or day 35 remained closed until week 12. First-closure ratio within 12 weeks was 75.6%. The median rate of newly developing wounds decreased significantly (P = 0.001) by 79.3%.ConclusionsComparison of the findings with published data from placebo arms and vehicle-treated wounds in controlled clinical trials suggests potential capability of ABCB5⁺ MSCs to facilitate wound closure, prolongate wound recurrence and decelerate formation of new wounds in RDEB. Beyond suggesting therapeutic efficacy for ABCB5⁺ MSCs, the analysis might stimulate researchers who develop therapies for RDEB and other skin fragility disorders to not only assess closure of preselected target wounds but pay attention to the patients’ dynamic and diverse overall wound presentation as well as to the durability of achieved wound closure and the development of new wounds.Trial registrationClinicaltrials.gov NCT03529877; EudraCT 2018-001009-98.     

Impact of chronicity of injury on the proportion of mesenchymal stromal cells derived from anterior cruciate ligaments

Background aimsThe graft-healing potential of mesenchymal stromal cells (MSCs) derived from the remnants of ruptured anterior cruciate ligaments (ACLs) after ACL reconstruction may depend on the chronicity of the injury. The aim of this study was to assess the quantitative and phenotypic differences between MSCs isolated from ACL remnants in patients with (sub)acute and chronic tearing.MethodsTorn ACL remnants were harvested during ACL reconstruction from 41 patients, 24 with (sub)acute ACL (<6 months from injury to surgery) and 17 with chronic ACL (time interval >6 months) tears. MSCs isolated from these samples were assessed for quantitative and phenotypic differences, and the correlation between the proportion of MSCs and the chronicity of ACL tear was evaluated.ResultsAt passage 0, the mean proportion of MSCs (CD34⁻, CD44⁺, CD90⁺ and CD105⁺) was higher in (sub)acute than in chronic ACL tear samples (20.69% ± 7.82% versus 9.85% ± 8.01%, P < 0.001). At passages 1 and 2, however, MSC proportions did not differ significantly in the two groups. Time interval showed a negative correlation with MSC proportion only at passage 0 (r = −0.505, P < 0.001). The optimal cutoff value for time from injury to surgery yielding <10% freshly isolated ACL-MSCs, a percentage expected to have low tissue healing potential, was 23.5 months.ConclusionsThe proportion of freshly isolated MSCs was higher in samples from patients with (sub)acute tearing than in chronic ACL tearing and negatively correlated with the time interval between trauma and surgery.     

Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System

BackgroundThe use of bone marrow–derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host disease, diabetes, ischemic cardiomyopathy and Crohn's disease, has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells must be cost-effective, safe and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures and require prolonged culture times.MethodsWe evaluated the Quantum Cell Expansion System for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results with a flask-based method currently in clinical trials.ResultsAfter only two passages, we were able to expand a mean of 6.6 × 10⁸ MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts and adipocytes. The Quantum was able to generate the target cell number of 2.0 × 10⁸ cells in an average of 9 fewer days and in half the number of passages required during flask-based expansion. We estimated that the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active.ConclusionsThe Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared with flasks. In the Quantum, the risk of contamination is substantially reduced because of the substantial decrease in open procedures.     

Is the subarachnoid administration of mesenchymal stromal cells a useful strategy to treat chronic brain damage?

Background aimsTraumatic brain injury (TBI) is a leading cause of mortality and morbidity worldwide. Developing effective protocols for the administration of mesenchymal stromal cells (MSCs) is a promising therapeutic strategy to treat TBI. It is important to develop alternatives to direct parenchymal injection at the injury site because direct injection is an expensive and invasive technique. Subarachnoid transplantation, a minimally invasive and low-risk procedure, may be an important and clinically applicable strategy. The aim of this study was to test the therapeutic effect of subarachnoid administration of MSCs on functional outcome 2 months after an experimental TBI in rats.MethodsTwo months after TBI, 30 female Wistar rats were divided into 3 groups (n = 10 in each group): sham, MSC (received 2 × 10⁶ MSCs) and saline (received only saline) groups. Neurological function, brain and spinal cords samples and cerebrospinal fluid were studied.ResultsNo significant differences were found in neurological evaluation and after histological analysis; differences in the expression of neurotrophins were present but were not statistically significant. MSCs survived in the host tissue, and some expressed neural markers.ConclusionsSimilar to direct parenchymal injections, transplanted MSCs survive, migrate to the injury cavity and differentiate into mature neural cell types for at least 6 months after engraftment. These results open the possibility that MSC administration through subarachnoid administration may be a treatment for the consequences of TBI. The transplantation technique and cell number should be adjusted to obtain functional outcome and neurotrophin production differences.     

Evaluation of an in vivo heterotopic model of osteogenic differentiation of equine bone marrow and muscle mesenchymal stem cells in fibrin glue scaffold

Autologous mesenchymal stem cells (MSCs) have been used as a potential cell-based therapy in various animal and human diseases. Their differentiation capacity makes them useful as a novel strategy in the treatment of tissue injury in which the healing process is compromised or delayed. In horses, bone healing is slow, taking a minimum of 6–12 months. The osteogenic capacity of equine bone marrow and muscle MSCs mixed with fibrin glue or phosphate-buffered saline (PBS) as a scaffold is assessed. Bone production by the following groups was compared: Group 1, bone marrow (BM) MSCs in fibrin glue; Group 2, muscle (M) MSCs in fibrin glue; Group 3, BM MSCs in PBS; Group 4, M MSCs in PBS and as a control; Group 5, fibrin glue without cells. BM and M MSCs underwent osteogenic stimulation for 48 h prior to being injected intramuscularly into nude mice. After 4 weeks, the mice were killed and muscle samples were collected and evaluated for bone formation and mineralization by using radiology, histochemistry and immunohistochemistry. Positive bone formation and mineralization were confirmed in Group 1 in nude mice based on calcium deposition and the presence of osteocalcin and collagen type I; in addition, a radiopaque area was observed on radiographs. However, no evidence of mineralization or bone formation was observed in Groups 2–5. In this animal model, equine BM MSCs mixed with fibrin glue showed better osteogenic differentiation capacity compared with BM MSCs in PBS and M MSCs in either carrier.     

Use of Real-Time PCR Technique in Studying Rumen Cellulolytic Bacteria Population as Affected by Level of Roughage in Swamp Buffalo

A real-time polymerase chain reaction approach was used in this study to determine the population of major ruminal bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) in digesta and rumen fluid of swamp buffalo (Bubalus bubalis). Four rumen-fistulated, male swamp buffalo were randomly assigned according to a 4 × 4 Latin square design to evaluate the effect of the urea-treated rice straw (roughage source)-to-concentrate ratio on cellulolytic bacterial distribution. Animals were fed roughage-to-concentrate (R:C) ratios of 100:0, 75:25, 50:50, and 25:75, respectively. At the end of each period, rumen fluid and digesta were collected at 0 h and 4 h post-morning-feeding. It was found that feeding urea-treated rice straw solely increased these three cellulolytic bacteria numbers up to 2.65 × 10⁹ and 3.54 × 10⁹ copies per milliliter for F. succinogenes, 5.10 × 10⁷ and 7.40 × 10⁷ copies per millilter for R. Flavefaciens, and 4.00 × 10⁶ and 6.00 × 10⁶ copies per milliliter for R. albus in rumen fluid and digesta, respectively. The distribution of the three cellulolytic bacteria species in digesta were highest at 3.21 × 10⁹, 4.55 × 10⁷, and 4.56 × 10⁶ copies per milliliter for F. succinogenes, R. flavefaciens, and R. albus, respectively. Moreover, at 4 h post-morning-feeding, the populations of the three cellulolytic bacteria were higher than found at 0 h post-morning-feeding. It is most notable that F. succinogenes were the highest in population in the rumen of swamp buffalo and cellulolytic bacteria mostly adhered to feed digesta in the rumen.     

In vivo safety profile and biodistribution of GMP-manufactured human skin-derived ABCB5-positive mesenchymal stromal cells for use in clinical trials

Background aimsHuman dermal ABCB5-expressing mesenchymal stromal cells (ABCB5⁺ MSCs) represent a promising candidate for stem cell–based therapy of various currently uncurable diseases in several fields of regenerative medicine. We have developed and validated a method to isolate, from human skin samples, and expand ABCB5⁺ MSCs that meet the guideline criteria of the International Society for Cellular Therapy. We are able to process these cells into a Good Manufacturing Practice–conforming, MSC-based advanced-therapy medicinal product.MethodsTo support the development of ABCB5⁺ MSCs for potential therapeutic topical, intramuscular and intravenous administration, we have tested our product in a series of Good Laboratory Practice–compliant nonclinical in-vivo studies addressing all relevant aspects of biosafety, including potential long-term persistence and proliferation, distribution to nontarget tissues, differentiation into undesired cell types, ectopic tissue formation, tumor formation and local tissue reaction.Results(i) Subcutaneous application of 1 × 10⁷ ABCB5⁺ MSCs/animal and intravenous application of 2 × 10⁶ ABCB5⁺ MSCs/animal, respectively, to immunocompromised mice did not result in safety-relevant biodistribution, persistence or proliferation of the cells; (ii) three monthly subcutaneous injections of ABCB5⁺ MSCs at doses ranging from 1 × 10⁵ to 1 × 10⁷ cells/animal and three biweekly intravenous injections of 2 × 10⁶ ABCB5⁺ MSCs/animal, respectively, to immunocompromised mice were nontoxic and revealed no tumorigenic potential; and (iii) intramuscular injection of 5 × 10⁶ ABCB5⁺ MSCs/animal to immunocompromised mice was locally well tolerated.DiscussionThe present preclinical in vivo data demonstrate the local and systemic safety and tolerability of a novel advanced-therapy medicinal product based on human skin-derived ABCB5⁺ MSCs.     

Mesenchymal stem cells can improve anal pressures after anal sphincter injury

Objective.Fecal incontinence reduces the quality of life of many women but has no long-term cure. Research on mesenchymal stem cell (MSC)-based therapies has shown promising results. The primary aim of this study was to evaluate functional recovery after treatment with MSCs in two animal models of anal sphincter injury.Methods.Seventy virgin female rats received a sphincterotomy (SP) to model episiotomy, a pudendal nerve crush (PNC) to model the nerve injuries of childbirth, a sham SP, or a sham PNC. Anal sphincter pressures and electromyography (EMG) were recorded after injury but before treatment and 10 days after injury. Twenty-four hours after injury, each animal received either 0.2 ml saline or 2 million MSCs labelled with green fluorescing protein (GFP) suspended in 0.2 ml saline, either intravenously (IV) into the tail vein or intramuscularly (IM) into the anal sphincter.Results.MSCs delivered IV after SP resulted in a significant increase in resting anal sphincter pressure and peak pressure, as well as anal sphincter EMG amplitude and frequency 10 days after injury. MSCs delivered IM after SP resulted in a significant increase in resting anal sphincter pressure and anal sphincter EMG frequency but not amplitude. There was no improvement in anal sphincter pressure or EMG with in animals receiving MSCs after PNC. GFP-labelled cells were not found near the external anal sphincter in MSC-treated animals after SP.Conclusion.MSC treatment resulted in significant improvement in anal pressures after SP but not after PNC, suggesting that MSCs could be utilized to facilitate recovery after anal sphincter injury.     

Endothelium-independent vasorelaxant effect of sodium ferulate on rat thoracic aorta

AimsThis study was designed to investigate the effects of sodium ferulate (SF) on rat isolated thoracic aortas and the possible mechanisms.Main methodsIsometric tension was recorded in response to drugs in organ bath. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured using Fluo-3 in cultured rat aortic smooth muscle cells (RASMC).Key findingsSF (0.1–30 mM) relaxed the isolated aortic rings precontracted with phenylephrine (PE) and high-K⁺ in a concentration-dependent manner with respective pD₂ of 2.7 ± 0.02 and 2.6 ± 0.06. Mechanical removal of endothelium did not significantly modify the SF-induced relaxation. In Ca²⁺-free solution, SF noticeably inhibited extracellular Ca²⁺-induced contraction in high-K⁺ and PE pre-challenged rings, and suppressed the transient contraction induced by PE and caffeine. The vasorelaxant effect of SF was unaffected by various K⁺ channel blockers such as tetraethylammonium, glibenclamide, 4-aminopyridine, and barium chloride. In addition, SF concentration-dependently reduced the contraction induced by phorbol-12-myristate-13-acetate, an activator of protein kinase C (PKC), in the absence of extracellular Ca²⁺, with the pD₂ of 2.9 ± 0.03. In RASMC, SF had no effect on PE- or KCl-induced [Ca²⁺]_i increase either in the presence or in the absence of external Ca²⁺.SignificanceThese results indicate that SF acts directly as a non-selective relaxant to vascular smooth muscle. The direct inhibition of the common pathway after [Ca²⁺]_i increase may account for the SF-induced relaxation in Ca²⁺-dependent contraction, while the blockage of the PKC-mediated contractile mechanism is likely responsible for the SF-induced relaxation in Ca²⁺-independent contraction.