"Turning Blood into Brain: Cells Bearing Neuronal Antigens Generated in Vivo from
Bone Marrow".

Éva Mezey ^1*, Karen J. Chandross ², Gyöngyi Harta ¹, Richard A. Maki ^{3, 4}, Scott R. McKercher ³

¹ Basic Neuroscience Program,
² Laboratory of Developmental Neurogenetics, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
³ The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
⁴ Neurocrine Biosciences, 10555 Science Center Drive, San Diego, CA 92121, USA.

* To whom correspondence should be addressed.  E-mail: mezey@codon.nih.gov

Bone marrow stem cells give rise to a variety of hematopoietic lineages and repopulate the blood throughout adult life. We show that, in a strain of mice incapable of developing cells of the myeloid and lymphoid lineages, transplanted adult bone marrow cells migrated into the brain and differentiated into cells that expressed neuron-specific antigens. These findings raise the possibility that bone marrow-derived cells may provide an alternative source of neurons in patients with neurodegenerative diseases or central nervous system injury.

 Mice homozygous for a mutation in the PU.1 gene were used as bone marrow transplant recipients. PU.1 is a member of the ETS (DNA binding domain) family of transcription factors and is expressed exclusively in cells of the hematopoietic lineage. In the absence of donor bone marrow cells, PU.1 knockout mice lack macrophages, neutrophils, mast cells, osteoclasts, and B and T cells at birth (15, 16). These animals are born alive but require a bone marrow transplant within 48 hours after birth to survive and develop normally. There are no gross morphological differences in the brain cytoarchitecture of these mice versus wild-type mice. In the present study, PU.1 null mice were used as bone marrow recipients to optimize the number of cells derived from the donor and to permit an accurate estimation of the numbers of bone marrow cells that migrate into the nervous system.

NeuN, a nuclear protein that is found exclusively in neurons (17-19), was used as a neuronal marker. Specific NeuN immunoreactivity was not present in acutely isolated (20) bone marrow cells. Acutely isolated bone marrow cells were also examined for neural antigens in our transgenic mouse line in which oligodendrocytes and Schwann cells express LacZ (21). No LacZ-expressing or b-galactosidase-immunopositive cells were present, and there was no specific immunostaining for NG2 chondroitin sulfate proteoglycan or O4, antigens that are present in Schwann cells and oligodendrocytes (22-24). These results strongly suggest that the bone marrow cell preparations were devoid of neurons and glia at the time of transplantation. When adult bone marrow cells were grown in culture for several weeks, the neural stem cell antigen, nestin, was present in 18% of the population [see Web fig. 1 (25)], indicating that bone marrow can give rise to neural stem cells.

Within 24 hours after birth, PU.1 homozygous recipients were given intraperitoneal injections of bone marrow cells from wild-type mice (20). Seven transplant recipient mice and nontransplanted control littermates were examined between 1 and 4 months of age. To determine the efficiency of the transplantation, we analyzed different organ tissues for the presence of donor-derived cells. Y chromosome-positive male cells were identified in hematopoietic organs of female recipients by fluorescent in situ hybridization histochemistry. Greater than 90% of spleen cells, in both white and red pulp, and ~10 to 15% of liver cells were Y chromosome-positive. All brains were examined by using a combination of in situ hybridization (ISH) to detect the Y chromosome and immunohistochemistry to visualize the neuronal nuclear marker, NeuN. Brains from a 4-month-old nontransplanted female [Fig. 1A and Web fig. 2, A to E (25)] and a nontransplanted male [Fig. 1B and Web fig. 2, F to J (25)] were processed together and served as controls for the Y chromosome hybridization specificity and efficiency (26). There was no specific Y chromosome staining in the female brain. The Y chromosome was frequently localized at the periphery of the nucleus, which is characteristic of heterochromatin (27, 28). The NeuN immunostaining was predominantly localized to the nucleus, although some neurons [as reported by others (19)] also exhibited perinuclear staining [Figs. 1 and 2 and Web figs. 2 to 5 (25)].

Marrow-derived cells (i.e., Y chromosome-positive) were present in the CNS of all of the transplanted mice examined. Between 2.3 and 4.6% of all cells (i.e., all identifiable nuclei, including vasculature) were Y chromosome-positive (Table 1). The Y chromosome-bearing cells were evenly distributed throughout the different brain regions [Fig. 1D and Web fig. 2, K and L (25)], in both white and gray matter. The Y chromosome was present in 0.3 to 2.3% of the NeuN-immunoreactive nuclei (Table 1). Confocal microscopy confirmed the presence of the Y chromosome in NeuN-immunopositive nuclei [Fig. 2 and Web figs. 4 and 5 (25)]. Y chromosome staining was localized to NeuN immunopositive cells and was not associated with any other neighboring nuclei in the x, y, or z planes. In the CNS of transplanted female mice, all NeuN-immunopositive nuclei were found in neuron-specific enolase (NSE)-containing cells (Fig. 1C). In the brain, NSE is expressed exclusively in neurons (29), demonstrating that Y chromosome-bearing cells can express two neuronal antigens. Most of these cells were found in the cerebral cortex [Web fig. 3, A to F (25)]; however, they were also present in the hypothalamus (Fig. 1, E to G), hippocampus, amygdala [Web fig. 3, G to I (25)], periaqueductal gray, and striatum. We did not detect Y chromosome-positive large motor neurons in the spinal cord or brainstem. A substantial number of Y chromosome-positive cell nuclei were present in cells within the choroid plexus of the lateral ventricle, in the ependyma of the ventricular system, and in the subarachnoid space, suggesting the cerebrospinal fluid as a primary route of entry [Web fig. 6 (25)]. We did not observe an overall increased density of Y chromosome-positive cell nuclei in neurogenic regions, including the subventricular zone, olfactory migratory region, or hippocampus. Because mesodermal stem cells can differentiate into microglia (8) and all microglia in these recipient animals arise from the donor bone marrow and are also Y chromosome-positive, we could not determine regional differences in the distribution of Y chromosome-positive nuclei.

These studies demonstrate that bone marrow cells migrate into the brain and differentiate into cells that express neuron-specific antigens. In combination with previous in vivo studies (9, 12, 13), the present work suggests that the bone marrow can supply the brain with an alternative source of neural cells. Neurons and macroglia (oligodendrocytes and astrocytes) are thought to arise from pluripotent neural stem cells that are present both in the developing (30) and adult mammalian CNS (31-35). It has been estimated that, for every 2000 existing neurons, one new neuron is produced each day (35, 36). In the rodent brain, there are two well-characterized neurogenic regions: one in the subgranular zone of the dentate gyrus and one in the forebrain subventricular zone (37-41). Two populations of neural stem cells have been identified in adult mammals: one in the ependymal cell layer lining the ventricles (33) and one in the subventricular zone [glial fibrillary acidic protein-immunoreactive cells (34), each of which gives rise to glial cells and neurons]. We suggest that, in addition to these sources of neural stem cells, there may be a continuous influx of bone marrow stem cells into the ependymal and subependymal zones that give rise to a variety of CNS neural cell types. An interesting possibility is that these entry routes might also serve as portals into the CNS for diseases that primarily originate in and affect the hematopoietic system (i.e., leukemia and AIDS).

 Bone marrow is far more accessible than neural stem cells and has the added advantage of having inherent host compatibility, thereby obviating the need to screen for viral and foreign antigens. Although our study showed that only a small number of transplanted cells expressed neuronal antigens in the adult brain, there may be factors that promote the differentiation of bone marrow cells into distinct neural cell types. Once these factors are identified, bone marrow cells might be expanded in vitro and provide an unlimited source of cells for the treatment of CNS disease and injury. Because at least two different types of stem cells have been isolated from bone marrow (hematopoietic and stromal), characterizing the potential for each population will be an important step toward optimizing regenerative therapies.

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   47. E.M. dedicates this report to the memory of János Szentágothai (1912-94), anatomist, statesman, romantic, artist, and mentor, who helped me understand the difference between looking at tissue sections and seeing the secrets they hold. The authors would like to express their sincere thanks to R. Dreyfus for his help with the conventional microscopy and C. L. Smith and R. Cohen for their help with the confocal microscopy. We are also grateful to M. Brownstein, R. Cohen, H. Gainer, L. Hudson, and M. Palkovits for their helpful suggestions and support throughout the work. These studies were supported by NIH grant AI30656 to R.A.M.

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