Conclusions And Future Directions

Since the late 1980s to early 1990s, a substantial body of data has been collected, clearly showing the promise of encapsulated cell therapy for treating a wide range of CNS disorders. Still, a number of research avenues exist that are incompletely explored and deserve attention prior to wide-scale clinical use of this technology. In some preclinical studies, the extent of diffusion from the implants appears to limit the therapeutic effectiveness of the encapsulated cells (62,68,69). Given the size of the human brain relative to the rodent and nonhuman primate brain, the potential problems related to limited tissue diffusion should be examined empirically. Studies in larger animals, such as nonhuman primates, should be conducted to include considerations of different numbers of devices and multiple reimplants distributed over long periods of time (>12 months). In this way, a reasonable assessment of the optimal spacing and distribution of multiple implants can be determined. This information could, at the same time, provide critical information regarding the relative risks of repeated tissue penetrations, tissue damage, and the potential for infection.

Encapsulation provides the opportunity to use cells from a variety of sources, including human and animal sources with and without genetic modification. In theory, the capsule should isolate the cells from the surrounding tissue. Still, if a capsule ruptures during implantation or retrieval, a deleterious

Figure 13 Neuronal cell counts in quinolinic acid-lesioned monkeys. Control-implanted animals displayed a significant loss of multiple types of striatal neurons including GABAergic (a and b), cholinergic (c and d), and diaphorase-positive neurons (e and f). Although neuronal loss was still present in animals receiving CNTF implants, it was significantly attenuated in both the caudate and putamen. In each case, data are presented as a percent of neurons on the lesioned/implanted side compared with the intact contralateral side. Representative photomicrographs for all 3 cell types are shown for both control and CNTF-implanted animals (a, GABAergic; c, cholinergic; and e, diaphorase-positive neurons) [see (99) for additional details].

Figure 13 Neuronal cell counts in quinolinic acid-lesioned monkeys. Control-implanted animals displayed a significant loss of multiple types of striatal neurons including GABAergic (a and b), cholinergic (c and d), and diaphorase-positive neurons (e and f). Although neuronal loss was still present in animals receiving CNTF implants, it was significantly attenuated in both the caudate and putamen. In each case, data are presented as a percent of neurons on the lesioned/implanted side compared with the intact contralateral side. Representative photomicrographs for all 3 cell types are shown for both control and CNTF-implanted animals (a, GABAergic; c, cholinergic; and e, diaphorase-positive neurons) [see (99) for additional details].

Figure 14 Photomicrographs of Nissl-stained sections through the striatum of monkeys that received quinolinic acid injections into the striatum followed by implants of encapsulated CNTF-producing (a) or control (b) BHK cells. A paucity of healthy neurons is observed in the striatum of control monkeys, which is in stark contrast to the numerous healthy appearing neurons seen in the same region of the CNTF-treated monkeys. Together with the sparing of strital neurons is a preservation of the GABE-ergic projection from the striatum to the globus pallidus. DARPP-32 immunocytochemistry revealed an intense, normal-appearing immunoreactivity within both the external and internal segments of the globus pallidus of CNTF-treated animals (c). In contrast, DARPP-32 immunoreactivity is reduced in control-implanted animals as a consequence of the lesion (d). Quantitative analysis confirmed the sparing of this projection in CNTF-treated monkeys.

Figure 14 Photomicrographs of Nissl-stained sections through the striatum of monkeys that received quinolinic acid injections into the striatum followed by implants of encapsulated CNTF-producing (a) or control (b) BHK cells. A paucity of healthy neurons is observed in the striatum of control monkeys, which is in stark contrast to the numerous healthy appearing neurons seen in the same region of the CNTF-treated monkeys. Together with the sparing of strital neurons is a preservation of the GABE-ergic projection from the striatum to the globus pallidus. DARPP-32 immunocytochemistry revealed an intense, normal-appearing immunoreactivity within both the external and internal segments of the globus pallidus of CNTF-treated animals (c). In contrast, DARPP-32 immunoreactivity is reduced in control-implanted animals as a consequence of the lesion (d). Quantitative analysis confirmed the sparing of this projection in CNTF-treated monkeys.

host immunological response could be induced. Although the host immune system should reject any released cells following capsule damage, the potential for tumorous growth remains a safety concern. Alterations in the ability of the host immune system to reject cells following damage to implants could also change upon long-term residence of the cells within the host. To date, no studies have systematically evaluated these risks, particularly with regard to the long-term effects of encapsulated cell implants. Again, primate studies using intact and intentionally damaged devices would provide a useful starting point for evaluating these issues. These studies could use normal and immunosuppressed animals, as well as evaluate potential tumorigenicity and changes in the host immune system over short and long periods of time.

Regulation of dosage is another area that deserves attention. In its most basic iteration, varying the numbers of cells within an implant, the size of the implant, or the use of multi ple implants, may permit a range of doses to be delivered. Although some long-term cell survival studies have been conducted (61-65), they have not systematically examined cell survival and output of the desired molecule over long periods of time. Rather, studies have provided a ''snapshot'' of survival and output at a single timepoint. Large, long-term, well-controlled studies need to be conducted to examine the relationship between variables that include time, cell survival, gene expression (when modified cells are used), neurochemi-cal output, the initial numbers of cell encapsulated, and the type of semipermeable membrane and extracellular matrix used for encapsulation. Obviously, such studies are time consuming and expensive. However, without them, the conditions optimal for successful cell encapsulation will remain speculative. It should be pointed out that some efforts are ongoing in this area, and a recent study raised the interesting possibility that dose control for dividing cells could be accomplished

Figure 15 The cells size distribution of layer V neurons in the motor cortex of monkeys that received striatal injections of quinolinic acid followed by implants of encapsulated BHK control (a,b) or CNTF-secreting BHK cells (c,d). This figure demonstrates that CNTF-producing grafts prevented the atrophy of cortical neurons that innervate the striatum. Note that in the control animals there is an increase in the number of neurons in the 0 to 100 ^m and 100 to 200 ^m range and a decrease in the number of neurons in the 300 to 400 and 400 to 500 ^m range ipsilateral to the lesion. This shift in cells size was not seen in those animals receiving CNTF-secreting cells.

Figure 15 The cells size distribution of layer V neurons in the motor cortex of monkeys that received striatal injections of quinolinic acid followed by implants of encapsulated BHK control (a,b) or CNTF-secreting BHK cells (c,d). This figure demonstrates that CNTF-producing grafts prevented the atrophy of cortical neurons that innervate the striatum. Note that in the control animals there is an increase in the number of neurons in the 0 to 100 ^m and 100 to 200 ^m range and a decrease in the number of neurons in the 300 to 400 and 400 to 500 ^m range ipsilateral to the lesion. This shift in cells size was not seen in those animals receiving CNTF-secreting cells.

with the use of cell-containing microcarriers in nonmitotic hydrogels (126).

Another area that has attracted little attention, concerns the variability in the in vivo performance of encapsulated cells and the possible role that the host tissue environment plays in this variability (51,66,67). As discussed earlier, it appears that at least some of the variability in device performance is attributable to differences between hosts. Although the mecha-nism(s) underlying these individual differences remain undetermined, several potential candidates exist, including the variations in the general health of the animals, between animal differences in immune function and undetected microbreaches in the polymer membrane prior to or during implantation. The notion that the viability of grafted cells may depend in part on host-related variability in the CNS environment has only been suggested for encapsulated cells to date. However, this emerging concept might also prove to be relevant for all CNS transplantation approaches that are cellular based. Indeed, the entire field of neural transplantation might benefit from this new perspective uncovered using encapsulated cells.

Finally, very few clinical studies have been conducted to date. Although several small safety studies have been completed, only one large, controlled clinical study has been performed using encapsulation technology. This study evaluated the use of encapsulated adrenal chromaffin cells for the treatment of pain but failed to reveal analgesia sufficient enough to continue the trials. As we have already discussed in a previous section, the selection of pain as an initial indication for detailed study might have been an unfortunate choice given that recent preclinical data using encapsulated chromaffin cells is mixed at best. The only other clinical targets under investigs-tion are ALS and HD, and these are apparently modest efforts. Until, larger, controlled clinical trials are conducted, the potential of this technology will not be fully realized.

In conclusion, it appears that the implantation of encapsulated cells may provide an effective means of alleviating the

Figure 16 Under local anesthesia, a device containing CNTF-secreting cells is implanted into the lumbar subarachnoid space via a small incision over the lumbar spine. A 19-gauge Touhy needle is inserted into the subarachnoid space. A flexible tip wire is inserted, the needle withdrawn, and a dilator passed through. A smaller cannula is then inserted over the wire and the cell-containing device is guided through the subarachnoid cannula. The silicone tether is secured to the lumbar fascia and the skin is sutured closed over the entire device.

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