• Infused human megakaryocytes release young platelets in the lungs with characteristics similar to donor platelets.• Platelets released from ex vivo-derived megakaryocytes are preactivated and compare poorly to donor platelets.Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo-generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application. (Blood. 2015;125(23):3627-3636)

IntroductionIn large-animal acute myocardial infarction (AMI) models, Wharton's jelly (umbilical cord matrix) mesenchymal stem cells (WJMSCs) effectively promote angiogenesis and drive functional myocardial regeneration. Human data are lacking.AimTo evaluate the feasibility and safety of a novel myocardial regeneration strategy using human WJMSCs as a unique, allogenic but immuno-privileged, off-the-shelf cellular therapeutic agent.Material and methodsThe inclusion criterion was first, large (LVEF ≤ 45%, CK-MB > 100 U/l) AMI with successful infarct-related artery primary percutaneous coronary intervention reperfusion (TIMI ≥ 2). Ten consecutive patients (age 32–65 years, peak hs-troponin T 17.3 ±9.1 ng/ml and peak CK-MB 533 ±89 U/l, sustained echo LVEF reduction to 37.6 ±2.6%, cMRI LVEF 40.3 ±2.7% and infarct size 20.1 ±2.8%) were enrolled.Results30 × 106 WJMSCs were administered (LAD/Cx/RCA in 6/3/1) per protocol at ≈ 5–7 days using a cell delivery-dedicated, coronary-non-occlusive method. No clinical symptoms or ECG signs of myocardial ischemia occurred. There was no epicardial flow or myocardial perfusion impairment (TIMI-3 in all; cTFC 45 ±8 vs. 44 ±9, p = 0.51), and no patient showed hs-troponin T elevation (0.92 ±0.29 ≤ 24 h before vs. 0.89 ±0.28 ≤ 24 h after; decrease, p = 0.04). One subject experienced, 2 days after cell transfer, a transient temperature rise (38.9°C); this was reactive to paracetamol with no sequel. No other adverse events and no significant arrhythmias (ECG Holter) occurred. Up to 12 months there was one new, non-index territory lethal AMI but no adverse events that might be attributable to WJMSC treatment.ConclusionsThis study demonstrated the feasibility and procedural safety of WJMSC use as off-the-shelf cellular therapy in human AMI and suggested further clinical safety of WJMSC cardiac transfer, providing a basis for randomized placebo-controlled endpoint-powered evaluation.

Stem cell therapy promises to become a minimally invasive method for the regeneration of the urethral rhabdosphincter muscle. Injecting a small number of cells does not preclude obtaining the desired therapeutic result.

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Mesenchymal Stem Cells (MSC) are employed in gene and cellular therapies. Routinely MSC are isolated from bone marrow mononuclear cells (MNC) by plastic adherence. Here we compared new isolation strategies of bone marrow MSC including immunodepletion of hematopoietic cells and immunomagnetic isolation of CD105 + and CD271 + populations. Four fractions were obtained: MNC MSC, RosetteSep-isolated MSC, CD105 + and CD271 + sorted MSC. We evaluated i) number of CFU-F colonies, ii) cell phenotype, iii) in vitro differentiation of expanded cells and iv) expression of osteo/adipogenesis related genes. Results: Average number of day 9 CFU-F colonies was the highest for CD271 positive fraction. Real-Time PCR analysis revealed expression of RUNX2, PPARγ and N-cadherin in isolated cells, particularly high in CD271 + cells. Expression of CD105, CD166, CD44, CD73 antigens was comparable for all expanded populations (over 90%). We observed various levels of hematopoietic contamination with the highest numbers of CD45 + cells in MNC-MSC fraction and the lowest in CD105 + and CD271 + fractions. Cells of all the fractions were CD34 antigen negative. Expanded CD105 and CD271 populations showed higher level of RUNX2, osteocalcin, PTHR, leptin, PPARγ2 and aggrecan1 genes except for α1 collagen. After osteogenic differentiation CD105 + and CD271 + populations showed lower expression of RUNX, PPARγ2 and also lower expression of osteocalcin and PTHR than MNC, with comparable α1-collagen expression. Chondrogenic and adipogenic gene expression was higher in MNC. More clonogenic CD105 + and particularly CD271 + cells, which seem to be the most homogenous fractions based on Real-Time PCR and immunostaining data, are better suited for MSC expansion.

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Interruption of spinal cord (SC) continuity leads to functional loss below the lesion level. The purpose of this study was to evaluate the safety and efficacy of bone marrow nucleated cell (BMNC) and multiple mesenchymal stem cell (MSC) transplantations in spinal cord injury (SCI). A patient with total SC interruption at the Th2-3 level underwent experimental therapy with BMNC and MSC transplantations followed with intensive neurorehabilitation treatment. At admission, 6 h after SCI, the patient was scored ASIA A, had a Th1 sensation level, paraplegia with sphincter palsy, and was without the ability to control trunk movement. Neurophysiology examination showed bilateral axonal damage to the motor and sensory neural fibers with no motor unit potentials or peripheral motor nerve conduction in the lower extremities. The standard therapy had been applied and had not produced any positive results. The patient was treated with autologous BMNCs injected intravenously (3.2 × 10 9 ) and intrathecally (0.5 × 10 9 ) 10 weeks after the SCI and with five rounds of MSCs every 3-4 months (1.3-3.65 × 10 7 ) administered via lumbar puncture. Total number of transplanted MSC cells during the course of treatment was 1.54 × 10 8 . There were no complications related to transplantations and no side effects related to the therapy during 2 years of treatment. The ASIA score improved from A to C/D (from 112 to 231 points). The sensation level expanded from Th1 to L3-4, and the patient's ability to control the body trunk was fully restored. Bladder filling sensation, bladder control, and anal sensation were also restored. Muscle strength in the left lower extremities improved from plegia to deep paresis (1 on the Lovett scale). The patient's ability to move lower extremities against gravity supported by the movements in quadriceps was restored. The patient gained the ability to stand in a standing frame and was able to walk with the support of hip and knee ortheses. Magnetic resonance imaging (MRI) revealed that at the Th2/Th3 level, where the hemorrhagic necrosis was initially observed, small tissue structures appeared. Our results suggest that repeated intrathecal infusions of MSCs might have the potential to produce clinically meaningful improvements for SCI patients.

There is a need among patients suffering from drug‐resistant epilepsy (DRE) for more efficient and less toxic treatments. The objective of the present study was to assess the safety, feasibility, and potential efficacy of autologous bone marrow cell transplantation in pediatric patients with DRE. Two females and two males (11 months to 6 years) were enrolled and underwent a combined therapy consisting of autologous bone marrow nucleated cells (BMNCs) transplantation (intrathecal: 0.5 × 109; intravenous: 0.38 × 109–1.72 × 109) followed by four rounds of intrathecal bone marrow mesenchymal stem cells (BMMSCs) transplantation (18.5 × 106–40 × 106) every 3 months. The BMMSCs used were a unique population derived from CD271‐positive cells. The neurological evaluation included magnetic resonance imaging, electroencephalography (EEG), and cognitive development assessment. The characteristics of BMMSCs were evaluated. Four intravenous and 20 intrathecal transplantations into the cerebrospinal fluid were performed. There were no adverse events, and the therapy was safe and feasible over 2 years of follow‐up. The therapy resulted in neurological and cognitive improvement in all patients, including a reduction in the number of epileptic seizures (from 10 per day to 1 per week) and an absence of status epilepticus episodes (from 4 per week to 0 per week). The number of discharges on the EEG evaluation was decreased, and cognitive improvement was noted with respect to reactions to light and sound, emotions, and motor function. An analysis of the BMMSCs' characteristics revealed the expression of neurotrophic, proangiogenic, and tissue remodeling factors, and the immunomodulatory potential. Our results demonstrate the safety and feasibility of BMNCs and BMMSCs transplantations and the considerable neurological and cognitive improvement in children with DRE. stem cells translational medicine 2018;7:20–33