A safe and effective magnetic labeling protocol for MRI-based tracking of human adult neural stem cells

Magnetic resonance imaging (MRI) provides a unique tool for in vivo visualization and tracking of stem cells in the brain. This is of particular importance when assessing safety of experimental cell treatments in the preclinical or clinical setup. Yet, specific imaging requires an efficient and non-perturbing cellular magnetic labeling which precludes adverse effects of the tag, e.g., the impact of iron-oxide-nanoparticles on the critical differentiation and integration processes of the respective stem cell population investigated. In this study we investigated the effects of very small superparamagnetic iron oxide particle (VSOP) labeling on viability, stemness, and neuronal differentiation potential of primary human adult neural stem cells (haNSCs). Cytoplasmic VSOP incorporation massively reduced the transverse relaxation time T2, an important parameter determining MR contrast. Cells retained cytoplasmic label for at least a month, indicating stable incorporation, a necessity for long-term imaging. Using a clinical 3T MRI, 1 × 103 haNSCs were visualized upon injection in a gel phantom, but detection limit was much lower (5 × 104 cells) in layer phantoms and using an imaging protocol feasible in a clinical scenario. Transcriptional analysis and fluorescence immunocytochemistry did not reveal a detrimental impact of VSOP labeling on important parameters of cellular physiology with cellular viability, stemness and neuronal differentiation potential remaining unaffected. This represents a pivotal prerequisite with respect to clinical application of this method

Density gradient centrifugation compromises bone marrow mononuclear cell yield

Bone marrow mononuclear cells (BMNCs) are widely used in regenerative medicine, but recent data suggests that the isolation of BMNCs by commonly used Ficoll-Paque density gradient centrifugation (DGC) causes significant cell loss and influences graft function. The objective of this study was to determine in an animal study whether and how Ficoll-Paque DGC affects the yield and composition of BMNCs compared to alternative isolation methods such as adjusted Percoll DGC or immunomagnetic separation of polymorphonuclear cells (PMNs). Each isolation procedure was confounded by a significant loss of BMNCs that was maximal after Ficoll-Paque DGC, moderate after adjusted Percoll DGC and least after immunomagnetic PMN depletion (25.6±5.8%, 51.5±2.3 and 72.3±6.7% recovery of total BMNCs in lysed bone marrow). Interestingly, proportions of BMNC subpopulations resembled those of lysed bone marrow indicating symmetric BMNC loss independent from the isolation protocol. Hematopoietic stem cell (HSC) content, determined by colony-forming units for granulocytes-macrophages (CFU-GM), was significantly reduced after Ficoll-Paque DGC compared to Percoll DGC and immunomagnetic PMN depletion. Finally, in a proof-of-concept study, we successfully applied the protocol for BMNC isolation by immunodepletion to fresh human bone marrow aspirates. Our findings indicate that the common method to isolate BMNCs in both preclinical and clinical research can be considerably improved by replacing Ficoll-Paque DGC with adapted Percoll DGC, or particularly by immunodepletion of PMNs

Anti-rat monoclonal antibodies used for flow cytometry.

<p>Anti-rat monoclonal antibodies used for flow cytometry.</p

Isolation of BMNCs from fresh human bone marrow aspirates and determination of progenitor cells.

<p>(A) Fresh whole bone marrow (BM) contained a high proportion of CD45- erythroid cells. The amount of erythroid cells could be significantly decreased by lysis or by immunodepletion (MACS or PluriB) of erythrocytes whereas highest purity of CD45+ leukocytes was attained after immunomagnetic depletion (MACS). (B) Lysis of whole bone marrow caused a significant loss of BMNCs (*p<0.05 versus whole bone marrow) that was further extended by PMN depletion with either MACS or PluriB (^#p<0.05 versus lysed bone marrow). The combined depletion of erythrocytes and PMNs by sequential MACS or by combined PluriB yielded comparable BMNC counts. The amount of remaining PMNs was constant at a low level after each of the isolation procedures. (C) Gating strategy for progenitor characterization. CD34+ hematopoietic stem cells (HSCs; C 1) featured a uniform, lymphoid-like phenotype and were partially CD133+ (C 3). In contrast, CD105+ mesenchymal stromal cells (MSC; C 2) exhibited increased variability of size and granularity. (D) Quantification of progenitors revealed that HSCs and MSCs were lost due to the different BMNC isolation procedures (p<0.05 versus lysed bone marrow). Cell loss was comparable in all experimental approaches except for the combined one-step depletion (PluriB), where the HSC yield was significantly lower compared to the other isolation protocols (^#p<0.05 versus lysis+MACS, lysis+PluriB and MACS+MACS). Values are means ± SD for 3 samples. *^,#p<0.05 by one-way ANOVA.</p

Representative illustration of the gating strategy in rat samples.

<p>The cells of interest were first categorized into cells belonging to the erythroid lineage (CD45−/Ery+), CD45−/Ery- cells and leukocytes (CD45+/Ery-). The latter were then differentiated into B-cells (CD45R+/CD11b−), CD11b+ cells (CD45+/CD45R−/CD11b+) and other CD45+ cells (CD45+/CD45R−/CD11b−). Next, CD11b+ cells were separated into RP1+ polymorphonuclear cells (PMN) and RP1- monocytes/dendritic cells (Mo/DC).</p

Rat BMNC yield following different isolation procedures.

<p>(A) Compared to lysed bone marrow, each of the isolation procedures caused an almost complete depletion of PMNs (C; arrows indicate the remaining RP1+ PMNs). As an unwanted side effect, this was accompanied by a significant loss of BMNCs and CD45- cells (A). BMNC loss was maximal after Ficoll DGC followed by Percoll DGC and MACS separation (*p<0.05). Both Percoll and MACS preserved the CD45- population compared to Ficoll (A; ^§p<0.05). Further analysis revealed a symmetric cell loss among the BMNC subpopulations (B; C, representative forward/sideward scatter diagrams). (D) For both Percoll and MACS, primarily RP1+ PMNs but also particular BMNC populations were detected within the waste (Percoll: layer 2; MACS: content of the columns). Values are means ± SD for 5 samples. *^,§p<0.05 by one-way ANOVA.</p

Influence of ammonium chloride lysis on rat bone marrow composition.

<p>(A) Freshly isolated rat bone marrow consists of 60% CD45+ and 40% CD45- cells. Lysis caused a significant decrease of CD45- (−85%) and CD45+ (−36%) cells. (B) The forward/sideward scatter diagram revealed an almost complete depletion of erythroid cells (arrow) whereas other main bone marrow populations remained largely unchanged. (C) Quantification of the cell fractions revealed that the loss of CD45+ cells due to lysis occurred symmetrically among the main leukocyte subpopulations. (D) Staining against an erythroid marker supported the finding that most cells of the erythroid line (CD45−/Ery+) disappeared following lysis whereas other CD45−/Ery- cells were significantly enriched. This was further potentiated after the application of Ficoll, Percoll or MACS depletion leading to an extended loss of erythroid cells in favor of CD45−/Ery-. Values are means ± SD for 5 samples. *p<0.05 by t-test.</p

Recovery of rat hematopoietic progenitors.

<p>(A) Analysis of the CFU-GM frequency revealed a significant enrichment of hematopoietic stem cells (HSC) by Ficoll compared to MACS. However, when relating the CFU-GM number to the absolute BMNC yield, Ficoll DGC resulted in a significant loss of HSCs compared to both Percoll and MACS separation. Values are means ± SD for 4 samples. *p<0.05 by one-way ANOVA.</p

Bone marrow cell transplantation time-dependently abolishes efficacy of granulocyte colony-stimulating factor after stroke in hypertensive rats

Background and Purpose— We aimed to determine a possible synergistic effect of granulocyte colony-stimulating factor (G-CSF) and bone marrow–derived mononuclear cells (BM MNC) after stroke in spontaneously hypertensive rats. Methods— Male spontaneously hypertensive rats were subjected to middle cerebral artery occlusion and randomly assigned to daily injection of 50 μg/kg G-CSF for 5 days starting 1 hour after stroke (groups 1, 2, and 3) with additional intravenous transplantation of 1.5×10E7 BM MNC per kilogram at 6 hours (group 2) or 48 hours (group 3) after stroke, or control treatment (group 4). Circulating leukocyte counts and functional deficits, infarct volume, and brain edema were repeatedly assessed in the first week and first month. Results— G-CSF treatment led to a significant neutrophilia, to a reversal of postischemic depression of circulating leukocytes, and to a significantly improved functional recovery without affecting the infarct volume or brain edema. BM MNC cotransplantation was neutral after 6 hours, but reversed the functional effect of G-CSF after 48 hours. Short-term investigation of combined G-CSF and BM MNC treatment at 48 hours indicated splenic accumulation of granulocytes and transplanted cells, accompanied by a significant rise of granulocytes in the circulation and the ischemic brain. Conclusions— G-CSF improved functional recovery in spontaneously hypertensive rats, but this effect was abolished by cotransplantation of BM MNC after 48 hours. In the spleen, transplanted cells may hinder the clearance of granulocytes that were massively increased by G-CSF. Increased circulation and infiltration of granulocytes into the ischemic brain may be detrimental for stroke outcome