Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.

The complex relationship between metabolic disease risk and body fat distribution in humans involves cellular characteristics which are specific to body fat compartments. Here we show depot-specific differences in the stromal vascual fraction of visceral and subcutaneous adipose tissue by performing single-cell RNA sequencing of tissue specimen from obese individuals. We characterize multiple immune cells, endothelial cells, fibroblasts, adipose and hematopoietic stem cell progenitors. Subpopulations of adipose-resident immune cells are metabolically active and associated with metabolic disease status and those include a population of potential dysfunctional CD8+ T cells expressing metallothioneins. We identify multiple types of adipocyte progenitors that are common across depots, including a subtype enriched in individuals with type 2 diabetes. Depot-specific analysis reveals a class of adipocyte progenitors unique to visceral adipose tissue, which shares common features with beige preadipocytes. Our human single-cell transcriptome atlas across fat depots provides a resource to dissect functional genomics of metabolic disease.

Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment.

Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous matrix deposition and prepare the construct for cartilage regeneration in situ. The chondrogenic capability of human BM-MSCs was first verified in a pellet culture. The BM-MSCs were then either seeded onto a composite scaffold rhCo-PLA combining polylactide and collagen type II (C2) or type III (C3), or commercial collagen type I/III membrane (CG). The BM-MSCs were either cultured in a proliferation medium or chondrogenic culture medium. Adult human chondrocytes (ACs) served as controls. After 3, 14, and 28 days, the constructs were analyzed with quantitative polymerase chain reaction and confocal microscopy and sulfated glycosaminoglycans (GAGs) were measured. The differentiated BM-MSCs entered a hypertrophic state by Day 14 of culture. The ACs showed dedifferentiation with no expression of chondrogenic genes and low amount of GAG. The CG membrane induced the highest expression levels of hypertrophic genes. The two different collagen types in composite scaffolds yielded similar results. Regardless of the biomaterial scaffold, culturing BM-MSCs in chondrogenic differentiation medium resulted in chondrocyte hypertrophy. Thus, caution for cell fate is required when designing cell-biomaterial constructs for cartilage regeneration.

The Utilization of Freezing Steps in Mesenchymal Stromal Cell (MSC) Manufacturing: Potential Impact on Quality and Cell Functionality Attributes.

Some recent reports suggest that cryopreserved and thawed mesenchymal stromal cells (MSCs) may have impaired functional properties as compared to freshly harvested MSCs from continuous cultures. A cryopreservation step in the manufacturing process brings important benefits, since it enables immediate off-the-shelf access to the products and a completion of all quality testing before batch release and administration to the patient. Cryopreservation is also inevitable in MSC banking strategies. In this study, we present the results from the MSC stability testing program of our in-house manufactured clinical-grade allogeneic bone marrow-derived MSC product that is expanded in platelet lysate and frozen in passage 2. The current manufacturing protocol contains only one freezing step and the frozen MSC product is thawed bed-side at the clinic. We can conclude superior viability and cell recovery of the frozen and thawed MSC product utilizing the validated freezing and thawing protocols we have developed. The MSC phenotype and differentiation potential was generally found to be unaltered after thawing, but the thawed cells exhibited a 50% reduced, but not completely abolished, performance in an in vitro immunosuppression assay. The in vitro immunosuppression assay results should, however, be interpreted with caution, since the chosen assay mainly measures one specific immunosuppressive mechanism of MSCs to suppress T-cell proliferation. Since at least two freezing steps are usually necessary in MSC banking strategies, we went on to investigate the impact of repeated freezing on MSC quality attributes. We can conclude that two freezing steps with a preceding cell culture phase of at least one passage before freezing is feasible and does not substantially affect basic cell manufacturing parameters or quality attributes of the final frozen and thawed product. Our results suggest, however, that an exhaustive number of freezing steps (≥4) may induce earlier senescence. In conclusion, our results support the utilization of frozen MSC products and MSC banking strategies, but emphasize the need of always performing detailed studies on also the cryopreserved MSC counterpart and to carefully report the cryopreservation and thawing protocols.

HLA-DR expression in clinical-grade bone marrow-derived multipotent mesenchymal stromal cells: a two-site study.

BACKGROUND: Contrary to the minimal criteria proposed by the International Society for Cell and Gene Therapy for defining multipotent mesenchymal stromal cells (MSC), human leukocyte antigen (HLA)-DR expression is largely unpredictable in ex vivo-expanded clinical-grade cultures. Although activation of MSC in culture does not appear to affect their functionality, a large study investigating the impact of HLA-DR expression on cell identity and potency is still missing in the literature. METHODS: A retrospective analysis of HLA-DR expression in 130 clinical batches of bone marrow (BM)-MSC from two independent Good Manufacturing Practice-compliant production facilities was performed in order to identify the consequences on critical quality attributes as well as potential activation cues and dynamics of MSC activation in culture. RESULTS: HLA-DR+ cells in culture were confirmed to maintain fibroblastic morphology, mesenchymal phenotype identity, multipotency in vitro, and immunomodulatory capacity. Interestingly, the use of either human sera or platelet lysate supplements resulted in similar results. CONCLUSIONS: HLA-DR expression should be considered informative rather than as a criterion to define MSC. Further work is still required to understand the impact of HLA-DR expression in the context of product specifications on BM-MSC qualities for clinical use in specific indications.

The use of unlicensed bone marrow-derived platelet lysate-expanded mesenchymal stromal cells in colitis: a pre-clinical study.

BACKGROUND: Mesenchymal stromal cells (MSCs) are a promising candidate for treatment of inflammatory disorders, but their efficacy in human inflammatory bowel diseases (IBDs) has been inconsistent. Comparing the results from various pre-clinical and clinical IBD studies is also challenging due to a large variation in study designs. METHODS: In this comparative pre-clinical study, we compared two administration routes and investigated the safety and feasibility of both fresh and cryopreserved platelet-lysate-expanded human bone marrow-derived MSCs without additional licensing in a dextran sodium sulfate (DSS) colitis mouse model both in the acute and regenerative phases of colitis. Body weight, macroscopic score for inflammation and colonic interleukin (IL)-1ß and tumor necrosis factor (TNF)α concentrations were determined in both phases of colitis. Additionally, histopathology was assessed and Il-1ß and Agtr1a messenger RNA (mRNA) levels and angiotensin-converting enzyme (ACE) protein levels were measured in the colon in the regenerative phase of colitis. RESULTS: Intravenously administered MSCs exhibited modest anti-inflammatory capacity in the acute phase of colitis by reducing IL-1ß protein levels in the inflamed colon. There were no clear improvements in mice treated with fresh or cryopreserved unlicensed MSCs according to weight monitoring results, histopathology and macroscopic score results. Pro-inflammatory ACE protein expression and shedding were reduced by cryopreserved MSCs in the colon. CONCLUSIONS: In conclusion, we observed a good safety profile for bone marrow-derived platelet lysate-expanded MSCs in a mouse pre-clinical colitis model, but the therapeutic effect of MSCs prepared without additional licensing (i.e. such as MSCs are administered in graft-versus-host disease) was modest in the chosen in vivo model system and limited to biochemical improvements in cytokines without a clear benefit in histopathology or body weight development.

Immunomonitoring of MSC-Treated GvHD Patients Reveals Only Moderate Potential for Response Prediction but Indicates Treatment Safety.

Mesenchymal stromal cells (MSCs) are used as salvage therapy to treat steroid-refractory acute graft-versus-host disease (aGvHD). We studied the immunological response to MSC treatment in 16 aGvHD patients by assessing lymphocyte profiles and three proposed aGvHD serum markers during the MSC treatment. Surprisingly, there were no obvious differences in the lymphocyte profiles between the responders and non-responders. The numbers of T, B, and NK cells were below the normal reference interval in all patients. CD4+ T helper (Th) cell levels remained particularly low throughout the follow-up period. The relative proportion of Th1 cells decreased, while regulatory T cells remained unaltered, and only very few Th2 and Th17 cells could be detected. Serum concentrations of regenerating islet-derived protein 3-alpha, cytokeratin-18 fragments (CK18F), and elafin were significantly elevated in patient samples compared with healthy controls, but only CK18F showed any potential in the prediction of patients' response to MSCs. No obvious markers for MSC therapy response were revealed in this study, but the results suggest that allogeneic MSCs do not provoke overt T cell-mediated immune responses at least in immunosuppressed aGvHD patients. The results advocate for the safety of MSC therapy and bring new insights in MSC immunomodulation mechanisms.

Recombinant human type II collagen hydrogel provides a xeno-free 3D micro-environment for chondrogenesis of human bone marrow-derived mesenchymal stromal cells.

Recombinant human type II collagen (rhCII) hydrogel was tested as a xeno-free micro-environment for the chondrogenesis of human bone marrow-derived mesenchymal stromal cells (BM-MSCs). The rhCII hydrogels were seeded with BM-MSCs and cultured in a xeno-free chondro-inductive medium for 14, 28 and 84 days. High-density pellet cultures served as controls. The samples were subjected to biochemical, histological and gene expression analyses. Although the cells deposited glycosaminoglycans into the extracellular space significantly more slowly in the rhCII hydrogels compared to the high-density pellets, a similar potential of matrix deposition was reached by the end of the 84-day culture. At day 28 of culture, the gene expression level for cartilage marker genes (i.e. genes encoding for Sox9 transcription factor, Collagen type II and Aggrecan) were considerably lower in the rhCII hydrogels than in the high-density pellets, but at the end of the 84-day culture period, all the cartilage marker genes analysed were expressed at a similar level. Interestingly, the expression of the matrix metallopeptidases (MMP)-13, MMP-14 and MMP-8, i.e. extracellular collagen network-degrading enzymes, were transiently upregulated in the rhCII hydrogel, indicating active matrix reorganization. This study demonstrated that the rhCII hydrogel functions as a xeno-free platform for BM-MSC chondrogenesis, although the process is delayed. The reversible catabolic reaction evoked by the rhCII hydrogel might be beneficial in graft integration in vivo and pinpoints the need to further explore the use of hydrogels containing recombinant extracellular matrix (ECM) proteins to induce the chondrogenesis of MSCs. Copyright © 2015 John Wiley & Sons, Ltd.

Expression of neural cell adhesion molecule and polysialic acid in human bone marrow-derived mesenchymal stromal cells.

BACKGROUND: In order to develop novel clinical applications and to gain insights into possible therapeutic mechanisms, detailed molecular characterization of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) is needed. Neural cell adhesion molecule (NCAM, CD56) is a transmembrane glycoprotein modulating cell-cell and cell-matrix interactions. An additional post-translational modification of NCAM is the α2,8-linked polysialic acid (polySia). Because of its background, NCAM is often considered a marker of neural lineage commitment. Generally, hBM-MSCs are considered to be devoid of NCAM expression, but more rigorous characterization is needed. METHODS: We have studied NCAM and polySia expression in five hBM-MSC lines at mRNA and protein levels. Cell surface localization was confirmed by immunofluorescence staining and expression frequency in the donor-specific lines by flow cytometry. For the detection of poorly immunogenic polySia, a fluorochrome-tagged catalytically defective enzyme was employed. RESULTS: All five known NCAM isoforms are expressed in these cells at mRNA level and the three main isoforms are present at protein level. Both polysialyltransferases, generally responsible for NCAM polysialylation, are expressed at mRNA level, but only very few cells express polySia at the cell surface. CONCLUSIONS: Our results underline the need for a careful control of methods and conditions in the characterization of MSCs. This study shows that, against the generally held view, clinical-grade hBM-MSCs do express NCAM. In contrast, although both polysialyltransferase genes are transcribed in these cells, very few express polySia at the cell surface. NCAM and polySia represent new candidate molecules for influencing MSC interactions.

Clumping and Viability of Bone Marrow Derived Mesenchymal Stromal Cells under Different Preparation Procedures: A Flow Cytometry-Based In Vitro Study.

Complications of microocclusions have been reported after intra-arterial delivery of mesenchymal stromal cells. Hence, quantification and efficient limitation of cell clumps in suspension before transplantation is important to reduce the risk. We used a flow cytometry-based pulse-width assay to assess the effects of different cell suspension concentrations (0.2-2.0 × 10(6)/mL), storage solutions (complete growth medium, Dulbecco's phosphate-buffered saline, and normal saline), storage time in suspension (0-9 h), and freeze-thawing procedure on the clumping of rat bone marrow derived mesenchymal stromal cells (BMMSCs) and also evaluated cell viability at the same time. Surprisingly, increasing the cell concentration did not result in more cell clumps in vitro. Freshly harvested (fresh) cells in normal saline had significantly fewer cell clumps and also displayed high viability (>90%). A time-dependent reduction in viability was observed for cells in all three storage solutions, without any significant change in the clumping tendency except for cells in medium. Fresh cells were more viable than their frozen-thawed counterparts, and fresh cells in normal saline had fewer cell clumps. In conclusion, cell clumping and viability could be affected by different cell preparation procedures, and quantification of cell clumping can be conducted using the flow cytometry-based pulse-width assay before intra-arterial cell delivery.

Rational Autologous Cell Sources For Therapy of Heart Failure - Vehicles and Targets For Gene and RNA Therapies.

This review focuses on the possibilities for intraoperative processing and isolation of autologous cells, particularly atrial appendage-derived cells (AADCs) and cellular micrografts, and their straightforward use in cell transplantation for heart failure therapy. We review the potential of autologous tissues to serve as sources for cell therapy and consider especially those tissues that are used in surgery but from which the excess is currently discarded as surgical waste. We compare the inculture expanded cells to the freshly isolated ones in terms of evidence-based cost-efficacy and their usability as gene- and RNA therapy vehicles. We also review how financial and authority-based decisions and restrictions sculpt the landscape for patients to participate in academic-based trials. Finally, we provide an insight example into AADCs isolation and processing for epicardial therapy during coronary artery bypass surgery.

A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells.

Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze-thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 10(9) ± 4.74 × 10(9) cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5-66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use.

Unexpected complication in a rat stroke model: exacerbation of secondary pathology in the thalamus by subacute intraarterial administration of human bone marrow-derived mesenchymal stem cells.

This study examined whether human bone marrow mesenchymal stromal/stem cells (BMMSCs) could alleviate the secondary pathology in the thalamus after middle cerebral artery occlusion (MCAO) in rats. Atypical accumulation of both amyloid-ß (Aß) and calcium in the thalamus was significantly higher in rats receiving the BMMSCs infusion 48 hours after MCAO as compared with the vehicle MCAO group. The elevated Aß/calcium accumulation correlated with the level of impaired sensorimotor function. Although secondary pathology in the thalamus seems to be rodent specific, it needs to be taken into account because it may impair long-term behavioral recovery and negate therapeutic treatment effects.

Safety and biodistribution study of bone marrow-derived mesenchymal stromal cells and mononuclear cells and the impact of the administration route in an intact porcine model.

BACKGROUND AIMS: Bone marrow mononuclear cells (BM-MNCs) and bone marrow-derived mesenchymal stem stromal cells (BM-MSCs) could have therapeutic potential for numerous conditions, including ischemia-related injury. Cells transplanted intravascularly may become entrapped in the lungs, which potentially decreases their therapeutic effect and increases the risk for embolism. METHODS: Twelve pigs were divided into groups of 3 and received (99m)Tc- hydroxymethyl-propylene-amine-oxime-labeled autologous BM-MNCs or allogeneic BM-MSCs by either intravenous (IV) or intra-arterial (IA) transplantation. A whole body scan and single photon emission computed tomography/computed tomography (SPECT/CT) were performed 8 h later, and tissue biopsies were collected for gamma counting. A helical CT scan was also performed on 4 pigs to detect possible pulmonary embolism, 2 after IV BM-MSC injection and 2 after saline injection. RESULTS: The transplantation route had a greater impact on the biodistribution of the BM-MSCs than the BM-MNCs. The BM-MNCs accumulated in the spleen and bones, irrespective of the administration route. The BM-MSCs had relatively higher uptake in the kidneys. The IA transplantation decreased the deposition of BM-MSCs in the lungs and increased uptake in other organs, especially in the liver. Lung atelectases were frequent due to mechanical ventilation and attracted transplanted cells. CT did not reveal any pulmonary embolism. CONCLUSIONS: Both administration routes were found to be safe, but iatrogenic atelectasis might be an issue when cells accumulate in the lungs. The IA administration is effective in avoiding pulmonary entrapment of BM-MSCs. The cell type and administration method both have a major impact on the acute homing.

Differential Clearance of Rat and Human Bone Marrow-Derived Mesenchymal Stem Cells From the Brain After Intra-arterial Infusion in Rats.

Intra-arterial (IA) delivery of bone marrow-derived mesenchymal stem cells (BM-MSCs) has shown potential as a minimally invasive therapeutic approach for stroke. The aim of the present study was to determine the whole-body biodistribution and clearance of technetium-99m ((99m)Tc)-labeled rat and human BM-MSCs after IA delivery in a rat model of transient middle cerebral artery occlusion (MCAO) using single-photon emission computed tomography (SPECT). Our hypothesis was that xenotransplantation has a major impact on the behavior of cells. Male RccHan:Wistar rats were subjected to sham operation or MCAO. Twenty-four hours after surgery, BM-MSCs (2 × 10(6) cells/animal) labeled with (99m)Tc were infused into the external carotid artery. Whole-body SPECT images were acquired 20 min, 3 h, and 6 h postinjection, after which rats were sacrificed, and organs were collected and weighed for measurement of radioactivity. The results showed that the majority of the cells were located in the brain and especially in the ipsilateral hemisphere immediately after cell infusion both in sham-operated and MCAO rats. This was followed by fast disappearance, particularly in the case of human cells. At the same time, the radioactivity signal increased in the spleen, kidney, and liver, the organs responsible for destroying cells. Further studies are needed to demonstrate whether differential cell behavior has any functional impact.

Xeno-free chondrogenesis of bone marrow mesenchymal stromal cells: towards clinical-grade chondrocyte production.

Current cell-based cartilage therapies relay on articular cartilage-derived autologous chondrocytes as a cell source, which possesses disadvantages, such as, donor site morbidity and dedifferentiation of chondrocytes during in vitro expansion. Due to these and other limitations, novel cell sources and production strategies are needed. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are a fascinating alternative, but they are not spontaneously capable of producing hyaline cartilage-like repair tissue in vivo. In vitro pre-differentiation of BM-MSCs could be used to produce chondrocytes for clinical applications. However, clinically compatible defined and xeno-free differentiation protocol is lacking. Hence, this study aimed to develop such chondrogenic differentiation medium for human BM-MSCs. We assessed the feasibility of the medium using three human BM-MSCs donors and validated the method by comparing BM-MSCs to three other cell types holding potential for articular cartilage repair. The effectiveness of the method was compared to conventional serum-free and commercially available chondrogenic differentiation media. The results show that the defined xeno-free differentiation medium is at least as efficient as conventionally used serum-free chondrogenic medium and performed significantly better on all cell types tested compared to the commercially available chondrogenic medium.

Extracellular o-linked N-acetylglucosamine is enriched in stem cells derived from human umbilical cord blood.

Stem cells have a unique ability to self-renew and differentiate into diverse cell types. Currently, stem cells from various sources are being explored as a promising new treatment for a variety of human diseases. A diverse set of functional and phenotypical markers are used in the characterization of specific therapeutic stem cell populations. The glycans on the stem cell surface respond rapidly to alterations in cellular state and signaling and are therefore ideal for identifying even minor changes in cell populations. Many stem cell markers are based on cell surface glycan epitopes including the widely used markers SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81. We have now discovered by mRNA analysis that a novel glycosyltranferase, epidermal growth factor (EGF) domain-specific O-linked GlcNAc transferase (EOGT), is highly expressed in stem cells. EOGT is responsible for adding O-linked N-acetylglucosamine (O-GlcNAc) to folded EGF domains on extracellular proteins, such as those on the Notch receptors. We were able to show by immunological assays that human umbilical cord blood-derived mesenchymal stromal cells display O-GlcNAc, the product of EOGT, and that O-GlcNAc is further elongated with galactose to form O-linked N-acetyllactosamine. We suggest that these novel glycans are involved in the fine tuning of Notch receptor signaling pathways in stem cells.

Human bone marrow mesenchymal stem/stromal cells produce efficient localization in the brain and enhanced angiogenesis after intra-arterial delivery in rats with cerebral ischemia, but this is not translated to behavioral recovery.

Intravascular cell therapy is a promising approach for the treatment of stroke. However, high accumulation of cells to lungs and other filtering organs is a major concern after intravenous (i.v.) cell transplantation. This can be circumvented by intra-arterial (i.a.) cell infusion, which improves homing of cells to the injured brain. We studied the effect of i.a. delivery of human bone marrow-derived mesenchymal cells (BMMSCs) on behavioral and histological outcome in rats after middle cerebral artery occlusion (MCAO). Sixty male Wistar rats were subjected to transient MCAO (60 min) or sham-operation. BMMSCs (1×10(6)) were infused into the external carotid artery on postoperative day 2 or 7. Histology performed after a 42-day follow-up did not detect any human cells (MAB1281) in the ischemic brain. Endothelial cell staining with RECA-1 revealed a significant increase in the number of blood vessels in the perilesional cortex in MCAO rats treated with cells on postoperative day 7. Behavioral recovery as assessed in three tests, sticky label, cylinder and Montoya's staircase, was not improved by human BMMSCs during the follow-up. In conclusion, human BMMSCs did not improve functional recovery in MCAO rats despite effective initial homing to the ischemic hemisphere and enhanced angiogenesis, when strict behavioral tests not affected by repeated testing and compensation were utilized.

Transient proteolytic modification of mesenchymal stromal cells increases lung clearance rate and targeting to injured tissue.

Systemic infusion of therapeutic cells would be the most practical and least invasive method of administration in many cellular therapies. One of the main obstacles especially in intravenous delivery of cells is a massive cell retention in the lungs, which impairs homing to the target tissue and may decrease the therapeutic outcome. In this study we showed that an alternative cell detachment of mesenchymal stromal/stem cells (MSCs) with pronase instead of trypsin significantly accelerated the lung clearance of the cells and, importantly, increased their targeting to an area of injury. Cell detachment with pronase transiently altered the MSC surface protein profile without compromising cell viability, multipotent cell characteristics, or immunomodulative and angiogenic potential. The transient modification of the cell surface protein profile was sufficient to produce effective changes in cell rolling behavior in vitro and, importantly, in the in vivo biodistribution of the cells in mouse, rat, and porcine models. In conclusion, pronase detachment could be used as a method to improve the MSC lung clearance and targeting in vivo. This may have a major impact on the bioavailability of MSCs in future therapeutic regimes.

Intra-arterial infusion of human bone marrow-derived mesenchymal stem cells results in transient localization in the brain after cerebral ischemia in rats.

Cell therapies from various sources have been under intense research in stroke. Efficient homing of the cells to the injured brain without complications is necessary to realize the therapeutic potential of cell therapy. Intra-arterial (IA) infusion of cells bypasses the filtering organs and directs the cells to the target area more efficiently. Here we studied the biodistribution of human bone marrow-derived mesenchymal stromal/stem cells (BMMSCs) after a direct infusion into the external carotid artery (ECA) in rats. Cells, which were cultured without animal-derived agents and also treated with a proteolytic enzyme to transiently modify cell surface adhesion proteins, were infused 24 h after transient middle cerebral artery occlusion (MCAO). SPECT imaging was used immediately after cell infusion and 24 h thereafter to track (111)In-oxine-labeled BMMSC in sham-operated and MCAO rats. IA infusion of BMMSCs in rats resulted in immediate cell entrapment in the brain, but the majority of the signal disappeared during the next 24 h and relocated to the internal organs. In MCAO rats, radioactivity counts 24 h after infusion were higher in the ischemic hemisphere compared to the contralateral hemisphere. Our results showed that IA infusion through ECA is a safe and efficient administration route for BMMSCs resulting in a transient localization of cells in the rat brain.

Cell surface structures influence lung clearance rate of systemically infused mesenchymal stromal cells.

The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM-MSCs) and umbilical cord blood (UCB-MSCs). The BM-MSCs and UCB-MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB-MSCs had higher expression levels of α4 integrin (CD49d, VLA-4), α6 integrin (CD49f, VLA-6), and the hepatocyte growth factor receptor (c-Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells.