Serotonin neurons derived from rhesus monkey embryonic stem cells: Similarities to CNS serotonin neurons

Ugur Salli, Arubala P. Reddy, Nurgul Salli, Nick Z. Lu, Hung Chih Kuo, Francis K.Y. Pau, Don P. Wolf, Cynthia L. Bethea

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

We sought an in vitro primate model for serotonin neurons. Rhesus monkey embryonic stem (ES) cell colonies were isolated and differentiated into embryoid bodies (EBs), then transferred to serum-free medium with 1% insulin-transferrin-selenium for 7 days to induce neural precursor cell (NPC) formation. NPCs were cultured in medium with 1% N-2 neural supplement and human fibroblast growth factor 2 (FGF2, 10 ng/ml) for 7 days to stimulate cell proliferation. Lastly, NPCs were dispersed into single cells and cultured without FGF2 for another 7 days to obtain terminal differentiation. Terminal cells were characterized for neuronal and serotonergic markers. Over 95% of the NPCs were immunopositive for nestin and Musashi1. Terminally differentiated cells appeared in both small and large morphologies. Most (>95%) of the mature cells (both small and large) were immunopositive for neuron-specific nuclear protein (NeuN), synaptophysin, microtubule-associated protein (MAP2C), Tau-1, neurofilament 160 (NF-160), beta-tubulin (TujIII), tryptophan hydroxylase (TPH), serotonin, the serotonin reuptake transporter (SERT), estrogen receptor-beta (ERβ), and progestin receptor (PR), but not estrogen receptor-alpha (ERα). Less than 2-3% of cells were positive for tyrosine hydroxylase (TH). Reverse transcriptase polymerase chain reaction (RT-PCR) detected mRNA transcripts for TPH-1, TPH-2, SERT, 5-HT1A- autoreceptor, ERβ, and PR in the differentiated population. A low level of expression of ERα mRNA was also detected. Quantitative RT-PCR indicated that the relative abundance of TPH-2 mRNA was greater than TPH-1 mRNA. Serotonin as measured by ELISA increased 3-fold in the mature stage compared to the selection and expansion stages. In summary, a remarkably high percentage of cells derived from monkey ES cells exhibited neuronal plus serotonergic markers as well as nuclear steroid receptors similar to primate CNS serotonin neurons, suggesting that these cells may serve as a useful primate model for serotonergic neurons.

Original languageEnglish (US)
Pages (from-to)351-364
Number of pages14
JournalExperimental Neurology
Volume188
Issue number2
DOIs
StatePublished - Aug 1 2004

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Embryonic Stem Cells
Macaca mulatta
Tryptophan Hydroxylase
Serotonin
Neurons
Estrogen Receptor beta
Fibroblast Growth Factor 2
Primates
Serotonin Plasma Membrane Transport Proteins
Messenger RNA
Progesterone Receptors
Reverse Transcriptase Polymerase Chain Reaction
Embryoid Bodies
Serotonergic Neurons
Nestin
Autoreceptors
Synaptophysin
Microtubule-Associated Proteins
Intermediate Filaments
Estrogen Receptor alpha

All Science Journal Classification (ASJC) codes

  • Neurology
  • Developmental Neuroscience

Cite this

Salli, Ugur ; Reddy, Arubala P. ; Salli, Nurgul ; Lu, Nick Z. ; Kuo, Hung Chih ; Pau, Francis K.Y. ; Wolf, Don P. ; Bethea, Cynthia L. / Serotonin neurons derived from rhesus monkey embryonic stem cells : Similarities to CNS serotonin neurons. In: Experimental Neurology. 2004 ; Vol. 188, No. 2. pp. 351-364.
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abstract = "We sought an in vitro primate model for serotonin neurons. Rhesus monkey embryonic stem (ES) cell colonies were isolated and differentiated into embryoid bodies (EBs), then transferred to serum-free medium with 1{\%} insulin-transferrin-selenium for 7 days to induce neural precursor cell (NPC) formation. NPCs were cultured in medium with 1{\%} N-2 neural supplement and human fibroblast growth factor 2 (FGF2, 10 ng/ml) for 7 days to stimulate cell proliferation. Lastly, NPCs were dispersed into single cells and cultured without FGF2 for another 7 days to obtain terminal differentiation. Terminal cells were characterized for neuronal and serotonergic markers. Over 95{\%} of the NPCs were immunopositive for nestin and Musashi1. Terminally differentiated cells appeared in both small and large morphologies. Most (>95{\%}) of the mature cells (both small and large) were immunopositive for neuron-specific nuclear protein (NeuN), synaptophysin, microtubule-associated protein (MAP2C), Tau-1, neurofilament 160 (NF-160), beta-tubulin (TujIII), tryptophan hydroxylase (TPH), serotonin, the serotonin reuptake transporter (SERT), estrogen receptor-beta (ERβ), and progestin receptor (PR), but not estrogen receptor-alpha (ERα). Less than 2-3{\%} of cells were positive for tyrosine hydroxylase (TH). Reverse transcriptase polymerase chain reaction (RT-PCR) detected mRNA transcripts for TPH-1, TPH-2, SERT, 5-HT1A- autoreceptor, ERβ, and PR in the differentiated population. A low level of expression of ERα mRNA was also detected. Quantitative RT-PCR indicated that the relative abundance of TPH-2 mRNA was greater than TPH-1 mRNA. Serotonin as measured by ELISA increased 3-fold in the mature stage compared to the selection and expansion stages. In summary, a remarkably high percentage of cells derived from monkey ES cells exhibited neuronal plus serotonergic markers as well as nuclear steroid receptors similar to primate CNS serotonin neurons, suggesting that these cells may serve as a useful primate model for serotonergic neurons.",
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Serotonin neurons derived from rhesus monkey embryonic stem cells : Similarities to CNS serotonin neurons. / Salli, Ugur; Reddy, Arubala P.; Salli, Nurgul; Lu, Nick Z.; Kuo, Hung Chih; Pau, Francis K.Y.; Wolf, Don P.; Bethea, Cynthia L.

In: Experimental Neurology, Vol. 188, No. 2, 01.08.2004, p. 351-364.

Research output: Contribution to journalArticle

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T1 - Serotonin neurons derived from rhesus monkey embryonic stem cells

T2 - Similarities to CNS serotonin neurons

AU - Salli, Ugur

AU - Reddy, Arubala P.

AU - Salli, Nurgul

AU - Lu, Nick Z.

AU - Kuo, Hung Chih

AU - Pau, Francis K.Y.

AU - Wolf, Don P.

AU - Bethea, Cynthia L.

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