Besides the identification of a novel astrocyte population that appears to have considerable promise as a cell-therapeutic tool to promote CNS injury repair, there are several other conclusions/hypotheses we can draw from these findings that we suggest have broader implications for the field of cell therapy as a whole and for neuroscience research in particular.
The first issue of concern is whether injured tissue can optimally induce the differentiation of transplanted precursor cells in a manner that best restores tissue function. Our data indicate there are significant reasons for thinking this is not always the case. In the specific instance of astrocyte generation in the injured spinal cord, the comparatively poor performance of GRP cells, as compared with GDAsBMP
, in promoting repair when transplanted into acute spinal cord injuries suggests that, in this instance, the injured cord is not inducing the generation of optimally useful populations of astrocytes. It is not yet known whether the reason GRP cells are less effective at promoting axon regeneration, protection of neurons, and recovery of locomotor function in SCI rats is due to an absence of factors in the injury site that are required to induce formation of astrocytes, such as GDAsBMP
, or whether precursor cells transplanted into acute spinal cord injuries are instead exposed to factors that induce their differentiation along alternative cell lineage pathways that result in cell types that are not supportive of spinal cord repair. In respect to the endogenous precursor cells of the spinal cord, the presence of Olig2+/GFAP+ and NG2+/GFAP+ astrocytes in the injury site [118
] suggests that these precursor cells are being induced to differentiate into cells that are more akin to GDAsCNTF
. Further support for this hypothesis is provided by studies from the Alonso lab [132
], indicating that endogenous adult glial progenitors contribute to scar formation at sites of injury via their differentiation into glial scar astrocytes.
If the endogenous precursor cells present in the adult tissue all represent specialized adult-specific type of O-2A progenitor cells [133
], it may be that they are only capable of differentiating into type-2 astrocytes, which have an antigenic phenotype resembling that of astrocytes generated from spinal cord GRP cells following exposure to CNTF or other gp130 agonists [40
Regardless of the specific reasons for the failure of GRP cells to provide the degree of benefit provided by GDAsBMP
, the benefits provided by transplantation of pre-differentiated GDAsBMP
have been robust in all of our studies [39
]. Therefore, this is a substantively different paradigm than applies to the repair of demyelinating damage for which it is progenitor cell transplantation that is required to obtain optimal repair, with transplantation of oligodendrocytes themselves being largely ineffective in this regard [136
In considering the previous issues, however, it is important to stress that we are at the earliest stages of understanding the complexity of the astrocyte-related response to CNS injury. For example, studies of Okada et al. [23
] have elegantly demonstrated important beneficial contributions of endogenous astrocytes (and/or their precursor cells) to the response to SCIs. Recent studies from the Kessler laboratory [24
] have shown that different BMP receptors (BMPR1a and BMPR1b) elicit different effects on gliosis, with BMPR1a promoting an early astrocytic hypertrophy response that appears to be beneficial. BMPR1b, in contrast, appears to be of primary importance in the later generation of reactive astrocytes that play an important role in glial scar formation and progression. Whether these two responses represent still another later of astrocyte complexity, or are related to the biology of the 2 astrocyte populations we have been studying is not yet known, but the possibility is intriguing to consider.
The second conclusion to emerge from our studies is that not all astrocytes are equivalent in their ability to promote repair. Functional heterogeneity in astrocyte populations has been observed for some time, both in vivo
and in vitro
]. It has not been known, however, whether such differences represent different traits of a single astrocyte population or truly distinct cell types generated from different progenitor cells and/or via different signaling mechanisms. In contrast, our studies unequivocally establish that different astrocytes are functionally very different in their effects on SCI repair. Our results further demonstrate that transplantation into the damaged CNS provides a useful approach to the discovery of functional differences between astrocyte populations.
A third lesson that emerges from our studies is that it is critically important to fully define cell populations (preferably at the clonal level) to pursue their development for tissue repair. GRP cells and O-2A progenitor cells are purified on the basis of antigenic phenotype, the timing of development at which they are isolated, and the tissue from which they are isolated, but the antibodies used for their purification are identical [25
]. Moreover, GRP cells can express the platelet-derived growth factor receptor-α, NG2 and Olig2, while still retaining their capacity to generate astrocytes with both type-1 and type-2 characteristics [78
] in vitro
. It is only by clonal analysis of GRP cells to be certain that one has a homogeneous population of such cells. If the field of cell therapy develops in a manner analogous to the development of pharmaceuticals, it will be increasingly important to demonstrate the absolute purity of the populations to be used for transplantation.
The previously described considerations suggest that too little is presently known to enable reliable generation of optimally beneficial astrocytes from human embryonic stem cells (ESCs) or from induced pluripotent stem cells. The means of preferentially generating GRP cells versus O-2A progenitor cells from either ESCs or induced pluripotent stem cells have not been identified, and the lack of antigenic markers that distinguish these populations means that it is not possible to directly purify either of these populations from induced cultures. Therefore, even though it is possible to generate astrocytes from ESCs, the types of astrocytes are not clear. It will be essential to compare such astrocytes with GDAsBMP to determine if they are equivalently useful in promoting repair.
The importance of attention to detail is indicated by the recent studies from Fischer et al., in which they also studied the effects of transplantation of human embryonic glial precursor cells and astrocytes generated from them by exposure to BMP [84
]. In agreement with our studies, no significant recovery in motor function was seen in animals receiving the human glial precursor cells. In sharp contrast with our studies, however, there was no significant recovery in motor function seen in the animals receiving the hGPC-derived astrocytes, and there was also no difference in the ability of either population to promote axonal outgrowth. What could be the explanation for the differences between their studies and those we conducted? Certainly, there were multiple differences in the approaches utilized in these studies as compared with our own. Fischer et al. carried out transplants into contusion injuries, in athymic rats, and delayed transplantation until 9 days after injury. They also grew cells on different substrates than used in our experiments. Our ongoing studies suggest that it is not these differences, however, that are the critical ones in respect to the study of regeneration. We note, however, that the use of athymic rats may have compromised the ability to adequately study neuropathic pain syndromes in these animals due to the importance of cells of the immune system in generating these syndromes [149
]. Finding out what differences are responsible for generating useful astrocytes versus
astrocytes that do not provide the robust benefits repeatedly seen in our own studies will be essential if we are to develop astrocyte transplantation therapies as a useful approach to CNS repair.