Additional file 1: of Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells

Detailed methods: lentiviral production and transduction, metabolic assays, and list of reagents for flow cytometry and PCR. Table S1. List of antibodies used for flow cytometry. Table S2. List of Taq-man assays used for qPCR. Table S3. Analysis of GO pathways shared in HIF-MSC vs GFP-MSC and MSC hypoxia vs MSC normoxia. Figure S1. Results from glycolytic activity of MSCs. (DOCX 130 kb

<i>Pten</i> ablation in DA cells affect the direct striatal output system by significantly increasing mRNA expression levels for DRD1 and pDyn in striatal medium spiny neurons.

<p>BDNF mRNA levels were significantly elevated in the ventral mesencephalon (A) Dopamie receptor D1 (DRD1) and D2 (DRD2) mRNA expression levels in the striatum of control (n = 9) and <i>Pten</i> KO animals (n = 13). DRD1 mRNA expression levels were significantly increased in KO animals as compared to controls. DRD2 mRNA levels were unchanged between the groups. (B) Changes in DRD1 expression levels were accompanied by an elevation in prodynorphin (pDyn) mRNA in <i>Pten</i> KO animals. Preproenkephalin (PPE) mRNA expression levels were unchanged. (C) BDNF mRNA levels were significantly increased in the ventral mesencephalon in KO animals. All data are mean±SEM. *p<0.05, Student's t-test.</p

<i>Pten</i> ablation shows neuroprotective effects in the intrastriatal 6OHDA mouse model.

<p>Animals were treated with a unilateral 6OHDA injection in the striatum, and perfused 4 weeks after the injection to analyze the effect of <i>Pten</i> ablation on the magnitude of the lesion. (A) The SNc from control animals (n = 4) was markedly affected by the lesion as reflected by the significant loss of TH positive profiles in the ipsilateral side to the lesion as compared to the contralateral side. The dopaminergic neurons in <i>Pten</i> KO (n = 6) animals were largely spared after the lesion when compared to control animals. (B) The neuroprotective effects related to <i>Pten</i> ablation were observed at the level of the striatal axonal projections as well. Depletion of TH positive terminals in the striatum was reduced across the striatum in control animals, while axon terminal degeneration was much lower in <i>Pten</i> KO animals. (C) Survival rates for TH-positive neurons were quantified by stereologic counts. The number of surviving neurons (presented as percent surviving neurons when compared to the side contralateral to the lesion) was significantly increased in <i>Pten</i> KO animals (n = 6) when compared to controls (n = 4). (D) Optical density measurement in the injected striatum revealed a nearly complete preservation of TH terminals in <i>Pten</i> KO animals. TH staining density was significantly lower in control 6OHDA-treated animals with the biggest difference observed in the medial portion of the striatum, in accordance with sterotaxic coordinated used for the lesion. (A) Scale bar, 250 µm and (B) 500 µm. All data are mean±SEM. *p<0.05, Student's t-test.</p

The morphological preservation of the <i>Pten</i> deficient nigrostriatal system after 6OHDA treatment correlates with functional recovery after the lesion.

<p>To determine the functionality of the nigrostriatal system after the lesion, we examined methamphetamine-induced rotations. Methamphetamine treatment increases the extracellular availability of endogenous DA in the striatum, and it causes animals with a partial DA depletion to rotate against the lesion side (ipsilateral), due to the imbalance in DA release between the striatae. Strong ipsilateral rotational behavior was observed in control animals (n = 8) after treatment with (+) methamphetamine HCl (2.5 mg/kg) at 14 and 28 days after the lesion. Ipsilateral rotational behavior was significantly reduced in <i>Pten</i> KO animals (n = 10). All data are mean±SEM. *p<0.05, Student's t-test.</p

Selective <i>Pten</i> deletion in DA cells results in unaltered extracellular dopamine dynamics in spite of a significant increase in tissue catecholamine content.

<p>(A) <i>Pten</i> KO mice showed no differences in basal extracellular dopamine as estimated by the x-intercept in the no net flux analysis. DA clearance was also unaltered as indicated by no change in the extraction fraction (slope of the no net flux regression line). (C) Depolarization evoked dopamine release was unaltered in <i>Pten</i> KO animals. The total tissue content of dopamine (DA) was increased in the dorsal striatum (B) and the midbrain (D) of <i>Pten</i> KO mice. Levels for the dopamine metablites DOPAC (dihydroxyphenylacetic acid), and HVA (homovanillic acid) were significantly inceased in the striatum and midbrain respectively. 3MT (3-methoxytyramine) levels remained unchanged as compared to controls. All data are mean±SEM. *p<0.05, Student's t-test.</p

Exposure to a novel environment does not enhance locomotor activity in <i>Pten</i> KO mice.

<p>Control (n = 11) and <i>Pten</i> KO (n = 15) animals were placed in activity chambers to measure the locomotor response to a novel environment. No significant differences were found in all parameters measured during a cummulative period of 30 minutes. Total distances traveled is shown as a representative measure. All data are mean±SEM. *p<0.05, Student's t-test.</p

The lack of PTEN in DA neurons results in a significant enlargement of the ventral midbrain area, attributable to an increase in the total number of DA neurons that are larger in size, and display more dendritic extensions.

<p>(A) The volume of the substantia nigra compacta (SNc), ventral tegmental area (VTA) and substantia nigra reticulata (SNr) was significantly larger in <i>Pten</i> KO animals (n = 4), as compared to controls (n = 4). The volume of all measured areas was at least 40% larger in KO animals, with the largest increment in size seen in the SNr. (B) The increase in volume of the SNc and VTA from <i>Pten</i> KO animals was accompanied by a significant increase in the number of TH positive neurons (B) and cell size (C) in both regions. (B) The mean number of TH positive profiles in <i>Pten</i> KO animals was 9401±105.1 in the SNc, and 4775±106.3 in the VTA, a 43% and 26% increase, respectively, when compared to controls. (C) The area of TH positive neurons was 47% and 39% larger in the SNc and VTA of <i>Pten</i> KO animals as compared to controls. (D) The increase in the total volume covered by the SNr was attributable to an increase in the number and caliber of TH-positive fibers present in <i>Pten</i> KO animals in parallel with an increase in the number and size of TH positive cells in the SNc and VTA. All data are mean±SEM. *p<0.05, Student's t-test.</p

Pten KO animals do not show differences in striatal TH fiber density measurements.

<p>(A, C) Optical density values did not reveal significant differences in striatal TH staining intensity between control (n = 4) and <i>Pten</i> KO animals (n = 4). (B) There were no significant differences in area measurements corresponding to rostral and medial striatum. A slight but significant increase in area was observed in the caudal striatal region of <i>Pten</i> KO animals as compared to controls. Scale bar, 500 µm. All data are mean±SEM. *p<0.05, Student's t-test.</p