Bone Marrow Derived Stem Cells as a Potential Cell Source for Neural Repair

Bone marrow cell transplantation into demyelinated (Sasaki et al., 2001; Akiyama et al., 2002a, 2002b; Inoue et al., 2003) or contused spinal cord (Hofstetter et al., 2002) has demonstrated remyelination and improved functional recovery, respectively. Moreover, transplantation of bone marrow cells into cerebral ischemia models has demonstrated reduced lesion size and improved functional outcome (Li et al., 2002; Iihoshi et al., 2004). The precise cell population in bone marrow responsible for these putative therapeutic effects is uncertain. However, bone marrow-derived stromal cells (MSCs) have been suggested to differentiate into bone, cartilage, cardiac myocytes, and neurons and glia both in vitro and in vivo. MSCs are thought to represent a very small proportion of cells in the mononuclear population of bone marrow, and a method to isolate and expand them in culture would provide a valuable tool to study their potential therapeutic efficacy in vivo.

Bone marrow cells can be enriched in MSCs by selecting for plastic-adherent cells. These stromal cells will grow to confluency in appropriate culture conditions as flattened fibroblast-like cells (Friedenstein, 1976). MSCs may be present in different proportions in the stromal cell fraction of various species. MSCs have a distinct cell surface antigen pattern including SH2+, SH3+, and CD34- (Majumdar et al., 1998). Methodologies have been established to culture human MSCs in very high purity. However, these cells had reduced mitogenic activity after about five cell doublings over the course of about 6 weeks. To generate larger numbers of human MSCs, they have been transfected with the human telomerase gene for immortalization (Kobune et al, 2003).

Autologous bone marrow cells isolated by density gradient (mononuclear layer) can remyelinate demyelinated spinal cord axons (X-EB lesion model) after either direct (Sasaki et al., 2001; Akiyama et al., 2002b) or intravenous (Akiyama et al., 2002a; Inoue et al. 2003) administration or direct microinjection into the lesion. Figure 7 shows myeli-nated profiles in the X-EB demyelinated spinal cord after intravenous delivery of mononuclear cells isolated from bone marrow. Many of the myelin profiles were SC-like. Several lines of evidence indicate that the remyelination following bone marrow cell transplantation was from the transplanted bone marrow cells and not from endogenous host cells. Our studies were carried out within 3 weeks after X-EB lesion induction, which is well within the time at which persistent demyelination is confirmed in a large number of control studies (Akiyama et al., 2002a, 2002b, 2004; Blakemore and Crang, 1985; Franklin et al., 1996; Honmou et al., 1996, Kato et al., 2000; Kohama et al., 2001; Sasaki et al., 2001). However, the possible recruitment of cells outside of the lesion zone by the injection procedure cannot be completely ruled out. We used acutely prepared autologous bone marrow mononuclear cells prepared on density gradient that were not expanded in culture and were therefore not able to incorporate reporter genes into the cells for more definitive assessment of the fate of the injected cells. However, sham injections of medium alone did not result in remyelination.

Figure 7 Remyelination in perivascular regions after intravenous injections of bone marrow. (A) A relatively large number of axons are remyelinated near a bed of capillaries. (B) A higher power micrograph shows endothelium of a capillary (arrowheads) and myelinated axon profiles near the capillary. Axons away from this capillary are not remyelinated (right). However, note that cells with dark nuclei are present in this nonmyelinated zone and some appear to be associated with axons, possibly in the process of remyelinating. (C) Longitudinal section through a capillary showing myelinated axons near the vessel, and a dark cell close to the capillary wall. (D) Histograms showing percentage of myelinated axons at various distances from vessel walls. Scale bar in C corresponds to 20 |m in A, 8 |m in B, and 5 |m in C. *P < 0.01 Analysis of variation (ANOVA). (Modified from Akiyama et al., 2002a.)

Figure 7 Remyelination in perivascular regions after intravenous injections of bone marrow. (A) A relatively large number of axons are remyelinated near a bed of capillaries. (B) A higher power micrograph shows endothelium of a capillary (arrowheads) and myelinated axon profiles near the capillary. Axons away from this capillary are not remyelinated (right). However, note that cells with dark nuclei are present in this nonmyelinated zone and some appear to be associated with axons, possibly in the process of remyelinating. (C) Longitudinal section through a capillary showing myelinated axons near the vessel, and a dark cell close to the capillary wall. (D) Histograms showing percentage of myelinated axons at various distances from vessel walls. Scale bar in C corresponds to 20 |m in A, 8 |m in B, and 5 |m in C. *P < 0.01 Analysis of variation (ANOVA). (Modified from Akiyama et al., 2002a.)

Moreover, the "dose-response" relationship of myelination with the number of injected cells further suggests that the donor cells were responsible for the remyelination (Inoue et al., 2003). Future studies with high-efficiency reporter gene incorporation into the endogenous cells will be critical to definitively distinguish between facilitation of endogenous repair vs. homing of injected cells into the lesion.

Intravenously delivered cells are unlikely to migrate across the blood-brain barrier (BBB) into normal spinal cord tissue, because the BBB would prevent cell access to the parenchyma. Indeed, we did not observe cells entering the nonlesion zone after intravenous delivery. Intravenously transplanted bone marrow cells appear to be recruited through the vascular system (Ferrari et al., 1998) and might be allowed to enter the spinal cord lesions because of the partial disruption of BBB in the X-EB lesions. In addition there may be active targeting mechanisms in the pathological environment of the lesion. Expression of chemotactic factors such as monocyte chemoattractant protein-1 is increased in damaged CNS tissues (Kim, 1996), and injured tissue extract selectively induces chemotaxis of mesenchy-mal progenitors in vitro (Chen et al., 2001). Adhesion molecules such as intercellular adhesion molecule (Zhang et al., 1995), vascular adhesion molecule-1, and E-selectin are also highly expressed on the endothelial cells in the damaged lesions (Blann et al., 1999; Haraldsen et al., 1996; Quesenberry and Becker, 1998; Zhang et al., 1995). Other unknown molecules may also promote migration of transplanted bone marrow cells into the demyelinated lesions. In addition, migration of bone marrow cells from the hematopoietic environment to the other nonhematopoietic tissues is associated with genetic transformation, in part through changes in expression of cell surface adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neural cell adhesion molecules (Haraldson et al., 1996; Quesenberry and Becker, 1998; Vermeulen et al., 1998). Thus, the interaction of these molecules may promote the transplanted bone marrow cells to target the spinal cord lesions.

The autologous bone marrow cells injected into the X-EB model rats were acutely prepared from the same demyeli-nated rats, with cells isolated by centrifugation through a density gradient (mononuclear cell layer) to remove ery-throcytes, platelets, and debris. The recovered mononuclear cell layer consisted of stromal cells, mesenchymal stem cells, hematopoietic and nonhematopoietic stem and precursor cells, and lymphocytes (Azizi et al., 1998). It remains unclear as to which type of cell within the bone marrow cell fraction is responsible for the in vivo differentiation of myelin-forming cells. Local injection of CD34+ cells (referred to as hematopoietic stem cells) did not result in remyelination in our demyelinated model system (Sasaki, et al., 2001), suggesting that hematopoietic stem cells are not a candidate cell for remyelination. MSCs have been reported to differentiate into neurons, astrocytes, and myelin-forming cells (Akiyama et al., 2002b; Woodbury et al., 2000), suggesting that cells within this fraction may be responsible for the myelin repair.

A large number of transplanted bone marrow cells into demyelinated lesions differentiated in vivo into myelin-form-ing cells (Inoue et al., 2003), although bone marrow cells predominantly differentiated into astrocytic and neuronal cell types in normal or ischemic brain (Eglitis et al., 1999). It is well established that stem cells from one region will, when placed in ectopic CNS sites, differentiate with a terminal phe-notype appropriate for that ectopic site (e.g., Akiyama et al., 2001; Brustle et al., 1999; Gage et al., 1995; Hammang et al., 1997; Snyder et al., 1997). The extracellular environment into which transplanted stem cells are delivered has an important influence on their fate in vivo (Akiyama et al., 2001; Brustle et al., 1999; Hammang et al., 1997; Snyder et al., 1997). Pathological CNS tissue is a different environment than intact CNS tissue and markedly alters the terminal differentiated phenotype of transplanted cells (Gage et al., 1995). The lesion into which the cells were placed in our model was enriched in axons because virtually all glia including astrocytes and oligodendrocytes were killed by the lesion protocol (Honmou et al., 1996). This abundance of axon membrane may be an important local environmental signal for bone marrow cell differentiation into myelin-forming cells. In the current demyelinated model, transplanted bone marrow cells retained the capacity to respond to local epigenetic signals in the demyelinated lesions, as they can differentiate into myelin-forming cell phenotypes.

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