3.1. Cell labeling and viability
The MRI detection of transplanted cells independently from the monitoring of pathology requires multi-nuclei MRI. Thus, a 19F-MRI cell labeling agent was employed. To ensure that cells retain their normal properties that are important to promote behavioral recovery, it is essential to establish an efficient labeling of cells without affecting cellular function. A time versus concentration curve of the bimodal 19F agent in two clinical-grade human neural stem cell lines, CTX0E03 and STROC05 () indicates that higher concentrations of agent over longer times in culture medium results in more uptake, as determined by fluorescence () and NMR spectroscopy (). After 6 and 24 hours of incubation, the CTX0E03 cells incorporate significantly more agent compared to the STROC05 cells. This label uptake highlights that not all neural stem cells will incorporate agent at the same rate. However, an efficient labeling of >97% of cells can be achieved () with agent being contained within the cell’s cytoplasm (). Importantly no significant amount of extracellular or membrane-bound agent can be observed using this approach.
Figure 1 Labeling of human neural stem cells with bimodal 19F agent. A. The human neural stem cell lines CTX0E03 and STROC05 were labeled with the 19F agent at different concentrations (1, 3, 5 mg/ml) for different amounts of time (3, 6, and 24h). Based on these (more ...)
3.2. Minor effects of 19F labeling on cell fate
Labeling of cells with the 19F agent resulted in a minor decrease in viability (4–12%). CTX0E03 cells exhibited a better tolerance to the labeling procedure compared to STROC05 cells. Labeling over 24 hours with 5 mg/ml of 19F agent did not result in a significant decrease of viability for the CTX0E03 () or STROC05 cells (). Based on the labeling efficiency and viability results, labeling of CTX0E03 with 5mg/ml for 24 hours was adapted as the standard protocol.
Figure 2 Effects of 19F on cell fate. Incorporation of the 19F agent had minimal effect on cell viability in CTX0E03 (A) and STROC05 cells (B) compared to control conditions (no contrast agent added to media). However, viability in STROC05 cells was more variable (more ...)
A cardinal feature of stem cells is their ability to proliferate and simultaneously produce more stem cells. This function was transiently affected in CTX0E03 cells within 24 hours of labeling as manifest by a 10% reduction in proliferating cells (). This was also reflected in mitochondrial activity (). Proliferation of CTXOE03 gradually decreased the amount of 19F agent present within a single cell, resulting in almost complete loss of detection after 4 days of proliferation (). Differentiating CTXOE03 labeled with the 19F agent also showed a decrease in label, but retained a higher amount after 7 days of differentiation (). It is important to note that differentiating will continue to dividefor a period of time before becoming post-mitotic. Cells retained their normal phenotypic characteristics () with nestin expression >90% in undifferentiated cells (). Although no significant effect of cell labeling was observed on neuronal differentiation, a significant decrease in astrocytic differentiation was evident.
The decrease of label in a cell population may be the result of cell divisions. Therefore, despite label retention within cells, a greater quantity of cells are needed for detection using the same amount of label. It is, however, also conceivable that some label is exocytosed from the cells (i.e. leakage) and is taken-up again by other cells. To evaluate this possibility, red 19F-labeled cells were co-cultured with green Cell Tracker-labeled cells (). At day 1 of co-culture, approximately 20% of cells were yellow indicating that some label transfer occurred. This could either be from live cells, from dead cells that contained the 19F agent or from extracellular agent that was not sufficiently cleared by washes. Over 7 days, there was a significant 10% increase in yellow cells. Therefore at least some transfer of the agent occurs over 7 days following in vitro labeling. The presence of transplanted cells needs to be carefully evaluated to determine to what degree this also occurs in vivo.
3.3. Establishing a cell detection threshold
An in vitro titration of 19F-labeled CTXOE03 cells was used to measure the 19F signal and contrast this with the signal for noise (i.e. 0 cells present). As expected, the 19F signal (and hence signal-to-noise) increased linearly with the number of cells (). A minimum of 1.7×104 cells/voxel is required for detection at the 1.25 SNR level. Therefore, a significant amount of cells is required within each voxel to allow detection. In the case of tissue engineering inside an infarct cavity caused by a stroke, a significant amount of cells is required to completely occupy the approximately 40 μl volume of lost tissue. Hence 19F MRI is appropriate for this application.
Figure 3 Detection threshold of 19F-labeled cells. Different concentrations (0, 1.5×106, 2.5×106, 4.5×106) of labeled cells were suspended in 6% gelatin to measure detection by 1H- and 19F-MRI. A weak 19F signal can be detected with 1.5×10 (more ...)
3.4. Non-invasive imaging of 19F-labeled cells and ECM bioscaffold
To enable tissue formation within the stroke cavity, the transplanted cells, however, require structural support and a compatible microenvironmental niche. As in normal tissue, such an environment can be provided by the ECM bioscaffold. Upon implantation into the stroke cavity the ECM+cells construct reduced the T2-hyperintensity that reflects the stroke pathology and also reduced the diffusion of water within the cavity (). Addition of CTXOE03 cells to the ECM bioscaffold did not further change the characteristics indicating that the ECM bioscaffold itself has the most dramatic effect upon the lesion cavity. Importantly, lack of tissue integrity of the ECM bioscaffold in the lesion cavity is still clearly visible on the apparent diffusion coefficient (ADC) maps, whereas boundaries between pathology and intact tissue on the T2-weighted image are more difficult to distinguish on these ex vivo MR images. Only transplanted cells labeled with the 19F agent produce a signal on the 19F MRI scan. Transplanted cells are distributed throughout the lesion cavity. However, especially anteriorly, small areas of infarction appear not to contain high levels of transplanted cells based on the 19F images ().
Figure 4 Ex vivo 19F- and diffusion-MR imaging. A. Middle cerebral artery occlusion (MCAo) induces a hyperintense signal on a T2-weighted MR image compared to a normal control. In the absence of 19F-labeled cells, no signal is detected in the 19F scan. The apparent (more ...)
A serial in vivo study further highlighted the potential of the ECM+cells construct to promote in situ tissue formation. 19F- and diffusion MRI are important tools to monitor this process. 19F-labeled cells target the lesion cavity based on stereotactic coordinates that are derived from pre-transplant MRI scans (). The injection tract is also visible 1 day following transplantation using 19F-MRI, but this is no longer visible 1 week post-transplantation. However, the presence of cells in the core of the lesion cavity is very clear, although the area covered by the 19F signal significantly decreased between 1 and 7 days. In one animal, dramatic changes in the posterior areas of the lesion cavity were apparent (). Pre-transplantation, a robust lesion cavity was visible on both the T2- and diffusion-MRI. After transplantation, some attenuation of this signal was apparent and based on the 19F MRI scan, transplanted cells were detected in the ventral part of the lesion. By 7 days following transplantation, the T2- and diffusion-MRI scans indicated a dramatically reduced infarction with transplanted cells still present within the ventral part of the lesion. These scans indicate that dramatic changes in the lesion cavity can be achieved by the transplantation of ECM bioscaffold seeded with human neural stem cells.
Figure 5 In vivo serial 19F- and diffusion-MR imaging. A. The distribution of 19F-labelled human neural stem cells within de-cellularised extra-cellular matrix encompass the lesion cavity. 19F-labelled cells can also be seen along the injection tract (green arrow) (more ...)
3.5. Histologic validation of 19F-MRI cell detection
As indicated by the 19F-MR images, at day 1 transplanted 19F-labeled CTXOE03 cells were contained within the injection tract. Immunohistochemistry validated this observation with labeled cells restricted to the injection tract as it penetrated through the tissue (). There was a good correspondence between the distribution of the 19F label and transplanted cells macroscopically, but at the cellular level it was evident that 19.2(±4)% of cells were host cells labeled with the 19F agent. It is unclear here if this was due to a transfer from live cells or a consequence of re-uptake from labeled dead cells also contained within the transplant. A small number of transplanted cells also did not contain detectable levels of the 19F agent. Similar observations were also found within the lesion cavity (), where the overall number and mass of transplanted cells was dramatically higher compared to the injection tract. As indicated by the 19F MR images, within the lesion cavity there were areas that are void of transplanted cells (). However, these areas contained the ECM bioscaffold material. Host cells can be seen to infiltrate the implanted ECM bioscaffold and there was some transfer of the 19F label to these host cells. Nevertheless, the agent does not diffuse out of the area of transplanted cells and therefore is a good marker of the topological distribution of these cells within the host brain.
Figure 6 Histological validation of the 19F-labeled cells. A. In the injection tract, 19F-labeled cells corresponded well to transplanted cells, but there was also a clear discrepancy between the 19F-label, transplanted and host cells. A fluorescent immunohistochemical (more ...)
3.6. ECM aids in situ tissue formation
Injection of the ECM bioscaffold filled the lesion cavity (), but small pockets of unfilled cavity remained, indicating that gelation and viscosity of ECM bioscaffold may require some optimization to achieve a complete and uniform filling of the cavity. Human neural stem cells (CTXOE03) mixed with the ECM bioscaffold prior to transplantation also exhibited a reasonably homogenous distribution, but cellular density was variable within the ECM bioscaffold(). Additionally, there were areas that were devoid of cells within the ECM bioscaffold (). Although host cells invaded the ECM bioscaffold, a better distribution of transplanted cells is desirable. In some locations, the ECM bioscaffold did not interface with the host tissue (). This is likely a consequence of ECM gelation of the ECM bioscaffold, which may have prevented homogeneous distribution or been affected by mixing with the extracellular fluid. It is important to note that the lesion cavity is a large volume to fill completely and homogenously. It is conceivable that surrounding host environment exerted some influence over the presence of cells within the ECM bioscaffold, as there was a clear posterior to anterior difference in cellular distribution and lesion coverage by the ECM bioscaffold and transplanted cells (). The ECM bioscaffold provided a structural support to the transplanted cells, and these cells mostly remained within the grafted area with excellent survival (). However, the cytoarchitecture of the de novo tissue is amorphous and distinct from normal striatal tissue. Very few transplanted cells were observed to migrate into areas of the ECM bioscaffold that attracted host cells (), and host cells appeared to preferentially migrate into areas of the ECM bioscaffold void of transplanted cells (). Host astrocytes infiltrate the ECM bioscaffold () and nested between grafted cells (), but very few transplanted cells differentiated into astrocytes. No neuronal differentiation of transplanted cells was observed. Cells infiltrating the ECM bioscaffold were predominantly of the microglia/macrophage phenotype (). This was evident in both ECM bioscaffold and ECM+cell transplants. Interestingly, microglia/macrophages were infrequently found in areas of transplanted cells but appeared to selectively infiltrate the unpopulated ECM bioscaffold.
Figure 7 Histological validation of de-cellularised extracellular matrix in stroke cavity. A. A trichrome staining reveals the area with the injected ECM as a dark pinkish area. The ECM injection mostly filled up the lesion cavity (yellow circle), but small areas (more ...)