27 January 2006. Researchers studying brain development have identified a key transcription factor that drives the differentiation of midbrain dopaminergic neurons, the cells that are lost in Parkinson disease. In a neat bit of reverse engineering, the investigators showed that introduction of the protein, the transcription factor Lmx1a, into mouse embryonic stem cells resulted in production of the same neurons in vitro. If the results, from Johan Ericson and colleagues in Stockholm and Paris, hold up in humans, this process offers the best chance yet to harness embryonic stem cell-derived neurons for transplantation to treat this disease.
Ericson’s results appear in today’s Cell, along with another important paper concerning the hematopoietic stem cells (HSCs) of adult bone marrow. In that work, Paul Frenette and his group at the Mount Sinai School of Medicine present the surprising finding that the sympathetic nervous system—best known for its role in the “fight or flight” response—regulates the release of HSCs from bone marrow into the circulation. The discovery reveals a fascinating and unsuspected link between the nervous system and blood and immune cell homeostasis. In addition, it raises new opportunities for the clinical manipulation of stem cell mobilization and homing, both important for the success of HSC transplantation.
For embryonic stem cell therapies, a major hurdle is enticing the cells to grow up into mature, and thus useful, citizens. What comes naturally to embryos—the guidance of ES cells down proper developmental pathways—remains a major challenge to researchers who hope to use in-vitro differentiated cells for transplantation therapies. For Parkinson disease, getting the right midbrain dopaminergic (DA) neurons from ES cells means choosing correctly one pathway out of a thousand that end up at that many different kinds of neurons. To find their way, Ericson and colleagues at the Karolinska Institute and the Ludwig Institute (both in Stockholm) and the Pasteur Institute in Paris, put their money on a bet that mapping the normal development of dopaminergic neurons would reward them with the key to generating neurons in vitro.
The effort, led by the three first authors Elisabet Andersson, Ulrika Tryggvason, and Qiaolin Deng, paid off in spades, as they were able to zero in on the homeobox domain transcription factor Lmx1a, and show that it was both necessary and sufficient to drive development of DA cells. By extensive PCR and in-situ hybridization studies on mouse embryos around the time that dopaminergic progenitors were forming, they found Lmx1a and another transcription factor, Msx1, to have the relevant pattern of expression. When they forced expression of Lmx1a in chick midbrain, the results were widespread ectopic formation of DA neurons. RNAi knockdown of Lmx1a in the same system caused a dramatic reduction in DA neuron production. Other results indicated that sonic hedgehog (Shh) signaling was required before Lmx1a expression, and that Msx1 acted downstream to complete the developmental program.
But the big question remained: Could these pathways be put to use to actually produce neurons in ES cell cultures? The answer was a resounding positive. When the researchers cultured mouse ES cells with Shh alone, they found many neurons, but none carried the DA marker, tyrosine hydroxylase (TH). But if they first transfected the cells with Lmx1a, the cultures quickly filled with TH+ neurons. A careful analysis of single cells showed they resembled bona fide midbrain DA neurons on the basis of coexpression of several other markers. The results suggest that exposing ES cells in culture to a combination of intrinsic (Lxm1a) and extrinsic (Shh) conditions may be the trick needed to generate the kinds of specialized neurons that could replace fetal tissue approaches and improve the outcome of transplantation therapy.
In the second paper, Frenette and colleagues set out to discover how hematopoietic stem cells (HSCs) are roused to leave the bone marrow and enter the blood stream. Clinically, treatment with G-CSF (granulocyte-colony stimulating factor) is commonly used to trigger HSC mobilization prior to harvest. The prevailing model holds that G-CSF affects local concentrations of the chemokine CXCL12, which is produced mainly by osteoblasts and attracts stem cells to bone. Lower CXCL12 concentrations favor release, but the details of its regulation were unclear.
Several years ago, Frenette et al. showed that the sulfated sugar polymer fucoidin could also mobilize stem cells, and he hypothesized that the compound might mimic an endogenous molecule, sulfated galactosylceramide, or sulfatide. To test the role of sulfatide (coincidentally, also a candidate biomarker for AD) in G-CSF-stimulated release, the researchers studied mice lacking UDP galactose:ceramide galactosyltransferase (Cgt), the enzyme that makes sulfatide and other galactocerebrosides. Indeed, the mice failed to mobilize stem cells in response to G-CSF, but not for the reason the investigators expected.
Galactocerebrosides are major components of myelin, and accordingly, the Cgt-null mouse suffers not from lack of sulfatide per se, but from faulty nerve transmission. In an extensive series of genetic and pharmacological experiments, the trio of first authors Yoshio Katayama, Michela Battista, and Wei-Ming Kao narrowed in on the sympathetic nervous system as a regulator of stem cell release. Their results established that rapid osteoblast suppression, down-regulation of CXCL12, and HSC release in response to G-CSF required the action of norepinephrine, the major neurotransmitter in the sympathetic nervous system. Consistent with this idea, treatment of mice with beta-blockers decreased HSC mobilization by G-CSF, and a beta agonist increased mobilization. Although the exact target of G-CSF action remains to be identified, results showed it acts in the peripheral nervous system.
The findings suggest novel ways to increase the harvest of HSC from blood, or to improve bone marrow engraftment after transplantation by manipulating the sympathetic nervous system. On a fundamental level, the unexpected demonstration that the nervous system plays a role in regulating the HSC niche fills in a gap in understanding how the system strikes a balance between different sites of hematopoiesis throughout the body. The work should also cause researchers to rethink their idea of what constitutes the HSC niche, and perhaps other niches as well, write Jonas Larsson and David Scadden of the Harvard Stem Cell Institute in Boston in an accompanying commentary. “Bone marrow, bone, and the nervous system now appear to integrate signals to regulate HSCs. If hematopoiesis is any guide, niches may be nodal points where multiple, previously disconnected systems collide,” they conclude.—Pat McCaffrey.
Andersson E, Tryggvason U, Deng Q, Friling S, Alekseenko Z, Robert B, Perlmann T, Ericson J. Identification of Intrinsic Determinants of Midbrain Dopamine Neurons. Cell. 2006 January 27; 124: 393-405. Abstract
Katayama Y, Battista M, Kao W, Hidalgo A, Peired AJ, Thomas SA, Frenette PS. Signals from the Sympathetic Nervous System Regulate Hematopoietic Stem Cell Egress from Bone Marrow. Cell. 2006 January 27; 124: 407-421. Abstract
Larsson J, Scadden D. Nervous Activity in a Stem Cell Niche. Cell. 2006 January 27; 124: 253-255. Abstract