MSCs Express Diverse Genes
To begin characterizing the undifferentiated MSCs more fully, we examined expression of a representative spectrum of genes that included those specific for diverse lineages. Our initial microarray screen of cultured dissociates revealed that the MSCs expressed many genes associated with differentiated cells. The expressed genes were not limited to mesodermal lineages but included genes representative of all germ layers. Listed in Table I are subsets of genes expressed by undifferentiated MSCs, adult tissue where they are highly expressed, and originating germ layer.
Table I. MSCs Express Diverse Genes Representative of All Germ Layers*
|Gene (accession number)||Symbol||Location||Layer|
|Lanosterol-14a-demethylase (U17697)|| ||Liver||Endoderm|
|IPP isomerase (AF003835)|| ||Liver||Endoderm|
|Leptin (D49653)|| ||Adipose||Mesoderm|
|Myosin heavy chain (L13606)|| ||Muscle||Mesoderm|
|NMDA receptor (S39221)|| ||Brain||Ectoderm|
|Ceruloplasmin (L33869)||CERU||Lung, Liver||Endoderm|
|SM22α (L41154)||SM22||Smooth muscle||Mesoderm|
|Protamine 2 (X14674)||PROT||Sperm||Germinal|
|Aldolase C (M63656)||ALDO||Brain||Ectoderm|
|Amyloid precursor protein (X07648)||APP||Brain||Ectoderm|
|NMDA glutamate binding subunit (S61973)||GLUT||Brain||Ectoderm|
To confirm this unexpected observation, we assayed transcript levels of some of these genes (bottom seven rows in Table I) by RT-PCR, focusing on prototypical genes specific for different germ layers. In addition to expression of expected mesodermal messages, such as SM22α, RT-PCR revealed mRNA for endodermal ceruloplasmin; ectodermal syntaxin 13, aldolase C, NMDA glutamate binding subunit, and amyloid precursor protein (APP); and germline protamine 2 (Fig. 1, Table 1). Omission of RTase in controls eliminated PCR products, confirming that the signals observed in the experimental groups were derived from RNA transcripts and not contaminating genomic DNA (Fig. 1).
Figure 1. Undifferentiated MSCs express genes representative of all germ layers. Population: RNA was harvested from primary cultures of undifferentiated MSCs and converted to cDNA, and target genes were amplified using specific primer pairs (see Table I for symbols). Amplification resulted in a single band in each reaction in which cDNA target was provided (RT +). Control lanes, in which no RTase was present during the cDNA synthesis reaction, did not yield product. Kilobase ladder (Kb) and λ/Hind III DNA (M) were employed as markers. Clonal: RNA was harvested from a clonally derived MSC population and RT-PCR performed as described above. A specific band was amplified in each reaction set.
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Gene Expression During Neuronal Differentiation
Although the expression of diverse gene products characteristic of distinct lineages by “undifferentiated” MSCs was unexpected, it may reflect extensive plasticity intrinsic to this stem cell population. Differentiation to specialized cell types, in turn, might be expected to alter differentially transcription of subsets of these messages. To examine this contention, we subjected MSCs to the neuronal differentiation protocol (Woodbury et al., 2000) and harvested RNA after 48 hr. Exposure to NIM caused a dramatic change in morphology of treated MSCs (Fig. 2). Before treatment, MSCs displayed a flat, fibroblastic morphology, with little evidence of refractility. At 48 hr posttreatment, MSC-derived neurons were rounded, exhibited highly refractile cell bodies, and displayed prominent process-like extensions. Neuronal differentiation significantly altered the pattern of gene expression of the foregoing prototypical target genes (Fig. 3). The transcription of germline protamine 2 was reduced to undetectable levels. Similarly, the expression of endodermal ceruloplasmin dramatically decreased. Perhaps unexpectedly, NMDA glutamate binding subunit mRNA levels also decreased. Expression of the neural genes aldolase C and syntaxin 13 exhibited modest decreases, as did muscle-specific SM22α. Among the genes surveyed, only APP expression remained unchanged as the MSCs assumed neuronal morphologic characteristics, allowing this signal to serve as an internal control, confirming that equal amounts of cDNA were present in each reaction.
Figure 2. Morphological differentiation of MSCs. Images of MSCs were captured before treatment (0 hrs) and after 48 hr (48 hrs) of exposure to NIM. MSCs rapidly transform from fibroblastic to neuronal morphologies. Images are representative of typical morphologies before and after treatment but are not from the same field.
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Figure 3. Neuronal differentiation of MSCs alters the pattern of gene expression. RNA was harvested from undifferentiated MSCs (S) or from MSC-derived neurons (N) 48 hr after neuronal induction. RT-PCR was performed to amplify gene products representing specific germ layers. Neuronal induction decreased expression of ceruloplasmin, NMDA glutamate binding subunit, and protamine 2 significantly, whereas expression of aldolase C, SM22α, and syntaxin was less affected. The level of expression of APP was not changed by neuronal induction. λ/Hind III DNA was used as a molecular weight marker (M).
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Expression of Neuroglial Genes
Having established the utility of RT-PCR in defining the unexpected expression of diverse differentiated genes in the “undifferentiated” MSCs and in neuronal differentiation, we examined transcriptional regulation of genes specific to the neuroglial lineage. APP served as a control, ensuring that equal amounts of target cDNA were used in each reaction (Fig. 4A). We examined glial fibrillary acidic protein (GFAP), the classical astrocyte marker (Eng et al., 1971), which is also expressed in neuroglial precursor cells (Laywell et al., 2000; Doetsch et al., 1999). Uncommitted MSCs expressed GFAP (Fig. 4A). With neuronal differentiation, the gene product was detectable at 24 hr, but at 48 hr postneuronal induction was no longer discernible, which is consistent with neuronal, but not glial, differentiation. Nevertheless, expression of GFAP by the MSCs is consistent with a growing body of evidence indicating that neural precursors in vivo express neuronal and glial markers and can differentiate to either lineage.
Figure 4. Neuronal differentiation alters the expression of precursor and mature neuronal messages. A: RT-PCR analysis was utilized to assess expression of neuroglial genes during the initial 48 hr of neuronal differentiation. APP expression in undifferentiated MSCs (S) and MSC-derived neurons at 24 hr (N 24) and 48 hr (N 48) postinduction remained constant. GFAP and neuroD (NeD) mRNA levels were elevated in MSCs and decreased as neuronal differentiation proceeded. Message for the neuronal markers NF-M and tau were undetectable in MSCs but increased with ongoing neuronal differentiation. Tau message was present in multiple isoforms (bracketed bands). λ/Hind III DNA served as an MW marker (M). B: RNA was harvested from undifferentiated MSCs maintained in SFM and from MSC-derived neurons maintained in NIM for 24 hr. RT-PCR performed with primers specific for NF-M yielded a single band from MSC-derived neurons. A very faint band corresponding to the NF-M product was also detected in cells maintained in SFM. RT-PCR of RNA derived from the cerebellum (Cb) of adult rats yielded an NF-M product of the same size, demonstrating specificity of the reaction. Equal amplification of noggin (inset) indicates equivalence of cDNA target. λ/Hind III DNA was used as an MW marker (M). C: RT-PCR followed by high-resolution electrophoresis revealed three distinct bands corresponding to known splice variants of tau message that were present in MSC-derived neurons at 48 hr postinduction (arrowheads). No signal was discernible in undifferentiated MSCs. Sizes (bp) of low-MW fragments of kilobase ladder (Kb) are indicated.
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To begin assessing mechanisms regulating neuronal differentiation, we examined neuroD, a transcription factor transiently expressed in neuronal precursor cells, known to regulate neuronal fate decisions (Lee, 1997; Morrow et al., 1999). Robust expression of neuroD was detected in the uncommitted MSCs, suggesting that these undifferentiated cells were already “primed” for neural differentiation (Fig. 4A). With differentiation, neuroD progressively decreased and was markedly diminished by 48 hr, which is consistent with transient expression of the transcription factor in differentiating neurons (Lee et al., 1995).
We used NF-M and tau as neuron-specific marker prototypes to examine changes in transcription associated with neuronal differentiation. We had previously found that the neuron-specific intermediate filament NF-M is expressed by MSC-derived neurons but not by undifferentiated MSCs (Woodbury et al., 2000). Here, we employed NF-M expression as a temporal, quantitative index of neuronal differentiation to help place the present observations in context. NF-M expression is associated with initiation of neuritogenesis, neural process outgrowth, and assumption of the characteristic mature neuronal morphology (Carden et al., 1987). Consistent with our previous findings, NF-M mRNA was undetectable in uncommitted MSCs but was present after 48 hr of neuronal induction. Similarly, tau message is undetectable in MSCs but is increasingly expressed with ongoing neuronal differentiation (Fig. 4A).
To examine further the changes in gene expression associated with neuronal differentiation, we investigated NF-M and tau expression in more detail. MSCs maintained in serum-free medium (SFM) for 24 hr exhibited extremely low, but detectable, levels of NF-M mRNA by RT-PCR, consistent with adoption of neuronal morphologies by a small number of cells under these conditions (Fig. 4B). However, incubation with the neural induction medium for 24 hr greatly enhanced NF-M message levels, consistent with differentiation of the vast majority of MSCs into neurons (Fig. 4B). As a positive control, we detected NF-M in the cerebellum of 32-day-old rats (Cb), generating a single band of the expected size. The encephalizing gene noggin (Smith and Harland, 1992) was unchanged 24 hr after neural differentiation, establishing the specificity of the increase in NF-M message.
Detailed analysis of the tau transcripts revealed that the known tau mRNA isoforms, generated by alternative splicing of exons 2 and 3 (Goedert et al., 1991), were present in the MSC-derived neurons but were not detectable in undifferentiated MSCs (Fig. 4C). Collectively, these data indicate that incubation in NIM decreases the expression of neural precursor messages (GFAP, neuroD) while simultaneously increasing the expression of specific neuronal markers (NF-M, tau), consistent with ongoing neuronal differentiation. In aggregate, our observations indicate that assumption of neuronal morphologies by MSCs is accompanied by a dramatic increase in the prototypical neuronal genes NF-M and tau, complex modulation of other neuronal genes, and decreased transcription of germline and mesodermal genes.
Tau was also detectable at the protein level by immunocytochemistry, confirming our PCR data. MSC-derived neurons, maintained in NIM for 10 days, were probed with antitau antibody. There was significant heterogeneity in the level of tau expression in the neurons even after 10 days, which often correlated with the degree of neuronal morphologic differentiation. For example, intensely tau-positive neurons (Fig. 5, arrow heads) with long varicose (Fig. 5, arrows) processes were evident, whereas neighboring cells exhibiting immature, transitional morphologic features displayed weak staining (Fig. 5A).
Figure 5. MSC-derived neurons express neuronal protein markers 10 days after induction. MSC-derived neurons were fixed and processed immunocytochemically for the expression of neuronal markers. A: An intensely tau-positive neuron (arrowhead) displays contracted cell body and long processes studded with varicosity-like swellings (arrows); neighboring cells lack both neuronal morphologies and strong tau staining. B: All MSC-derived neurons stained for β-III-tubulin expression. C: Synaptophysin is detected throughout the cell body, with intense staining in the varicosity-like swellings (arrows). inset: RT-PCR for synaptophysin was performed on RNA harvested from undifferentiated MSCs (S) and MSC-derived neurons 24 (N24) and 48 (N48) hr postinduction. A signal indicating the presence of synaptophysin message is not detected in MSCs but becomes increasingly evident with progressing neuronal differentiation. D: No staining is seen when the primary antibody is omitted from the reaction. E: Cells exhibiting neuronal morphologies show staining for ChAT (arrow heads), whereas flat stromal-like cells (arrow) do not. A perinuclear staining pattern is evident. F: A subpopulation of cells expresses TH (arrowheads), whereas most MSC-derived neurons (arrows) do not express this protein. Magnification ×200. Images in B–F were acquired through a blue filter to enhance contrast.
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We examined additional neuronal gene products that are expressed in more mature neurons or that are associated with functional neuronal communication. At 10 days, β-III tubulin, an intermediate filament characteristic of mature neurons (Menezes and Luskin, 1994), was present in virtually all cells (Fig. 5B). In contrast to these neuronal markers, MBP, a marker for mature oligodendrocytes, was not detectable in MSC-derived neurons (data not shown).
Genes Associated With Neurotransmission
To begin assessing the developing ability for functional communication, we initially examined synaptophysin, which is associated with synaptic vesicles and transmission. Analysis by RT-PCR indicated that synaptophysin mRNA was not present in undifferentiated MSCs but was detectable after 24 hr of neuronal differentiation and continued to increase thereafter (Fig. 5C, inset). The protein was detected in cell bodies as well as varicose, putative transmitter release sites along processes, reflecting an immature pattern of distribution (Fig. 5C). Control reactions in which the primary antibody was omitted were devoid of staining (Fig. 5D).
To assess further the developing capability for communicative function, we examined the expression of neurotransmitter enzymes. At 10 days, a large population of the neurons expressed choline acetyltransferase (ChAT), which catalyzes the synthesis of the excitatory transmitter acetylcholine (Fig. 5E). A similar percentage of ChAT-positive cells was observed when a monoclonal ChAT antibody from a different commercial source was used for staining, validating this staining pattern (data not shown). Interestingly, the majority of neurons (>85%) derived from the multipotent P19 embryonal carcinoma cell line are also cholinergic (Parnas and Linial, 1995). A smaller subpopulation of MSC-derived neurons expressed tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of catecholamines, dopamine, norepinephrine, and epinephrine (Fig. 5F).
Clonal Analysis of Derived Neurons
To assess further the differentiation potential of MSCs, we investigated the expression of neuronal markers in a clonal population derived from a single MSC. We had reported previously that clonal MSCs subjected to the differentiation protocol display neuronal morphologies and express high levels of NSE (Woodbury et al., 2000). We demonstrate here that clonal MSC-derived neurons expressed the neuronal markers tau (Fig. 6A), TOAD-64 (Fig. 6B), β-III tubulin (Fig. 6C), and ChAT (Fig. 6D). The arrowhead in Figure 6C indicates a β-III-tubulin+ MSC-derived neuron, and the arrow identifies the nucleus of an unstained MSC. Similarly, a ChAT+ MSC-derived neuron and an unstained nonneuronal cell are identified by an arrowhead and arrow, respectively in Figure 6D. In aggregate, these observations indicate that the MSC-derived neurons were developing the structural apparatus for synaptic communication and the machinery for transmitter signal biosynthesis. The expression of enzymes for different transmitters suggests that the MSC-derived neurons are capable of differentiating into multiple subtypes.
Figure 6. Clonally derived MSCs exhibit neuronal or osteoblastic phenotypes after differentiation. Neuronal differentiation of clonally derived MSCs express tau (A) and TOAD-64. (B) C: A β-III tubulin-positive MSC-derived neurons is indicated by the arrowhead. Arrow identifies the nucleus of an unstained cell. D: Most clonal MSC-derived neurons express ChAT (arrowhead). Arrow indicates an unstained cell not displaying neuronal morphologies. Magnification ×250. E,F: von Kossa staining of clonally derived MSCs maintained in osteogenic media for 13 days. Dark staining of the nodules demonstrates deposition of mineralized matrix characteristic of bone formation.
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To assess the ability of this clonal line to differentiate into mesenchymal derivatives, we grew these MSCs in osteogenic media for 13 days and probed for deposition of mineralized matrix. Under these conditions MSCs displayed cuboidal morphologies consistent with osteoblastic differentiation. Nodules of differentiated cells formed, producing mineralized matrix as demonstrated by von Kossa staining (Fig. 6E,F).
Morphologic Reversion and Gene Expression
Neuronal differentiation of MSCs exhibited notable plasticity at its earliest stages, when it was partially reversible. Cells exposed to NIM assumed characteristic neuronal morphologies, displaying refractile cell bodies and long processes by 24 hr postinduction (Fig. 7A). Withdrawal of NIM from MSC-derived neurons elicited process retraction and reversion of morphology within 24 hr to flat cells, which shared characteristics with uncommitted MSCs but generally displayed a more stellate morphology (Fig. 7B). To assess changes in gene expression associated with reversion, MSCs were differentiated to neurons by exposure to NIM for 24 hr. At this time, NIM was removed from half of the cells and replaced with SFM while the remaining cells were maintained in NIM. Twenty-four hours later (48 hr in total), we harvested RNA from neuronal and reverted cells and assessed changes in gene expression by RT-PCR. We again assayed expression of the archetypal targets from various germ layers expressed at high levels in uncommitted MSCs. Reversion from the neuronal to the MSC phenotype was associated with striking changes in expression of a subset of the target genes. Germline protamine 2 mRNA, which decreased dramatically with neuronal conversion, was highly expressed in the reverted MSCs. Similarly, endodermal ceruloplasmin message was detectable in MSCs, whereas depressed levels existed in the neurons. Strikingly, neuroD mRNA was highly expressed in the reverted MSCs, presumably indicating the potential for redifferentiation into neurons. GFAP message was also expressed, consistent with maintenance of a primitive neuroglial precursor state. In sum, the reverted MSCs appear to express multipotentiality, consistent with the plasticity displayed at the time of differentiation. In contrast, expression of several genes was unchanged by reversion to MSCs; message levels for APP, muscle-specific SM22α (Fig. 7C), syntaxin, and aldolase C (data not shown) remained the same.
Figure 7. Neuronal differentiation is reversible morphologically and transcriptionally. A: MSC-derived neurons at 24 hr postinduction display neuronal morphologies with highly refractile cell bodies and long process-like extensions. B: Same cells as in A 24 hr after NIM withdrawl. Cell bodies have flattened, and processes have been withdrawn, yielding cells with stromal morphologies. Magnification ×200. C: Morphologic reversion is accompanied by changes in gene expression, identified by RT-PCR. Ceruloplasmin, protamine 2, GFAP, and neuroD are expressed at higher levels in reverted cells (R 48) than in MSC-derived neurons (N 48) at 48 hr postinduction. The level of expression of APP and SM22α is unaltered by the reversion process.
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