Blood. 2006 March 1; 107(5): 2170–2179.

Figure 2. Expression of Ezh2 in hematopoietic cells. (A) To isolate the different lineage-negative (Lin-) populations from BM, the 5% most Lin- cells were selected. (B) Cells in the Lin- population were sorted based on Sca-1 and c-kit surface markers. (C) Expression of Ezh2 as measured by qPCR in the different Sca-1 and c-kit populations relative to Sca-1-c-kit- BM cells. (D) Growth of Lin-Sca-1+c-kit+ cells cultured in the presence of GM-CSF and SCF. (E) Relative expression of Ezh2 was monitored by qPCR at different time points after initiation of differentiation with GM-CSF and SCF. Day 0 was set at 1. Insert shows gel analysis of amplified RT-PCR products.

Blood. 2006 March 1; 107(5): 2170–2179.

Figure 3. Overexpression of Ezh2 in HSCs. (A) WBC counts of mice given transplants with BM cells transduced with control () or Ezh2 () vectors (n = 11 recipients per group, 2 independent experiments). Mean values plus or minus standard error of the mean (SEM) are shown. (B) Chimerism at different time points after transplantation. The percentage of donor-derived transduced CD45.1+GFP+ WBCs is shown. Average values ± 1 SEM of 2 independent experiments are shown. (C) Ezh2 protein expression in the spleen about 120 days after primary transplantation of control or Ezh2 CD45.1 BM cells. (D) CAFC frequencies of sorted LSK GFP+ cells 120 days after primary transplantation (control, ; Ezh2, ). (E) Chimerism levels of secondary recipients, competitively transplanted with various ratios (2:1, ⋄; 1:1, ○; 1:2, □; 1:5, ▵) of transduced/nontransduced and freshly isolated BM cells, analyzed 3 months after transplantation. Values show data from individual recipients in 2 independent experiments (n = 35/group). (F) CRI calculated for CD45.1+GFP+ (transduced) versus CD45.1+GFP- (nontransduced) cells (see insert). Values (+ 1 SEM) are averages 3 months after secondary transplantation, based on 11 and 24 individual mice in the first and second experiment, respectively (control, ; Ezh2, ). (G) CRI calculated for transduced cells (CD45.1+GFP+) compared to freshly isolated BM cells (CD45.2+; see insert). Averages values (+ 1 SEM) of 11 and 24 individual mice of the first and second experiment, respectively, are shown (control, ; Ezh2, ). (H) LTRA frequencies in CD45.1+GFP+ (transduced) cell fractions calculated from limiting dilution analyses 3 months after secondary transplantation in 2 independent experiments (control, ; Ezh2, ).

Blood. 2005 September 1; 106(5): 1574–1580.

Hematopoiesis in adults is believed to originate primarily from hematopoietic stem cells (HSCs) that reside within the bone marrow.1 Rare circulating forms of these stem cells are also detectable in the peripheral blood where they are speculated to traffic between the bone marrow and other organ compartments.2-4 In occasional disease states, bone marrow failure may lead to extramedullary hematopoiesis or the production of blood lineages from solid organs, such as the liver, that are normally considered nonhematopoietic.5-9

Blood. 2005 September 1; 106(5): 1574–1580.

Here we provide evidence that stem cells exist in adult murine liver that possess potent hematopoietic-reconstitution activity comparable with that of highly purified bone marrow–derived stem cells. The cells appear to be phenotypically distinct from normal bone marrow SP cells, yet may be bone-marrow derived.

Blood. 2005 September 1; 106(5): 1574–1580.

Overall, based on the relatively weak in vivo reconstitution activity reported for all purified organ-derived stem-cell populations, it has not been possible to determine whether organ-derived stem cells in general possess functional properties distinct from those of bone marrow–derived cells or if the methods used to date for the purification of those cells do not result in efficient purification of the relevant cells.

Blood. 2005 September 1; 106(5): 1574–1580.

MiceSingle-cell suspensions for FACS analysis and RNA extraction were prepared from 8- to 10-week-old female C57BL/6j mice. Transplantation studies used 8-week-old male donor C57BL/6-Ly5.1 (CD45.1; B6.SJL-Ptprca Pep3b/BoyJ) or C57BL/6-GFP mice that constitutively and ubiquitously express the enhanced green fluorescent protein (GFP) reporter gene under regulatory control of the chicken beta-actin promoter with cytomegalovirus enhancer (C57BL/6j-Tg(ACTB-EGFP)1Osb/J). Recipient mice were congenic 8- to 10-week-old female C57BL/6-Ly5.2 (CD45.2). All mice were obtained from Jackson Labs (Bar Harbor, ME) and maintained in a pathogen-free animal facility. Animal studies were approved by the Standing Committee on Animals of Harvard Medical School.

Blood. 2005 September 1; 106(5): 1574–1580.

Flow cytometryCell sorting was performed on a MoFlo triple laser instrument (DakoCytomation, Fort Collins, CO) using Summit 3.1 software (Dako Cytomation). Analysis of raw data was completed with FlowJo software (Treestar, Ashland, OR). The laser emissions were 488, 350, and 647 nm. Fluorescence was detected with the following bandpass filters: 530/40 for FITC and GFP, 580/30 for PE, 670/30 for PI, 405/30 for Hoechst blue, 570/20 for Hoechst red, and 670/20 for APC (Omega Optical, Brattleboro, VT). Hoechst blue and red fluorescence were detected in linear-scale acquisition. First, a live cell gate was created excluding cell fragments (low forward scatter) or events that contained high PI fluorescence. Cells within this live cell gate were displayed on a Hoechst-red–Hoechst-blue histogram, and side population (SP) cells were identified after collecting at least 1 × 105 events (gating algorithm illustrated in Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article).

Blood. 2005 September 1; 106(5): 1574–1580.

Reverse-transcription–polymerase chain reaction (RT-PCR)Total RNA was extracted from 8500 cells sorted from each bone marrow or liver population (Absolutely RNA Nanoprep Kit; Stratagene, La Jolla, CA). DNase treatment of all RNA extracts was included according to the manufacturer's instructions. cDNA was prepared by reverse transcription under standard conditions using random hexamer priming (First-Strand cDNA Synthesis Kit; Amersham Biosciences). PCR amplification of c-kit and glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA was performed using FastStart Taq DNA Polymerase (Roche) and the following primer pairs: c-kit forward, 5′-GCACTTGAGTGCTACACTCTTGCACCT-3′; c-kit reverse, 5′-TCTTCAGAACTGTCAACAGTTGGACAACA-3′; GAPDH forward, 5′-TTCCAGTATGACTCCACTCACG-3′; and GAPDH reverse, 5′-GTTCACACCCATCACAAACATG-3′. Conventional PCR conditions using one primer pair per reaction tube were as follows: 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, 40 cycles. Multiplex PCR to amplify both genes in the same reaction tube was performed under identical conditions with both primer pairs added to the same PCR reaction tube.

Blood. 2005 September 1; 106(5): 1574–1580.

Statistical analysisAverage blood chimerism was compared between groups by 2-tailed unpaired Student t test. A difference was considered statistically significant if P was less than .05.

Blood. 2005 September 1; 106(5): 1574–1580.

Figure 1. Adult liver and bone marrow SP cells and identification of the liver CD45+ SP tip. FACS analysis of Hoechst-stained single-cell suspensions identifies a side population (SP) of cells in murine adult liver (A), Ficolled adult liver (B), and Ficolled bone marrow (C). Closer inspection of the dot plot of liver SP shown in panel B reveals a cluster of SP cells with highly efficient dye-efflux properties (termed SP tip cells) that is distinct from cells found higher in the SP gate (SP shoulder cells; gates from B are redrawn in D to show cell frequencies of tip and shoulder subpopulations). Hoechst dye efflux in SP cells is inhibited by verapamil (E). Frequencies of cells expressing the panhematopoietic surface marker, CD45, in liver SP tip, shoulder, and non-SP cells are shown in panels F, G, and H, respectively. Rare CD45+ cells, representing just 3% of SP tip cells, are identified in panel F. Numbers in all graphs represent percentage of events contained in the illustrated gate.

Ann Gen Psychiatry. 2007; 6: 25.

Background Type-1 diabetes mellitus (DM) is a lifelong serious condition which often renders the application of standard treatment options for patients' comorbid conditions, such as bipolar disorder I, risky – especially for acute manic episodes. We present such a case whereby the application of standard anti-manic treatments would have jeopardized a patient whose physical condition was already compromised by DM.

Ann Gen Psychiatry. 2007; 6: 25.

MethodsWe report the case of a 55-year-old female with a history of type-1 DM since the age of 11, and severe ocular and renal vascular complications thereof. While on the waiting list for pancreatic islet cell transplantation, she developed a manic episode that proved recalcitrant to a treatment with gabapentin, lorazepam and quetiapine. Moreover, her mental state affected adversely her already compromised glycemic control, requiring her psychiatric hospitalization. Her psychotropic medication was almost discontinued and replaced by oxcarbazepine (OXC) up to 1800 mg/day for 10 days.

Ann Gen Psychiatry. 2007; 6: 25.

Type-1 diabetes mellitus (DM) is a lifelong condition of glycemic metabolism, with devastating and life-threatening systemic complications. Furthermore, the overall lifelong management of type-1 DM raises crucial issues of patients' compliance to strict dietary and pharmacological regimens as well as their capacity for resilience in the face of chronic psychosocial stressors. Moreover, treatment options of patients' comorbid conditions are often restricted, as their side effects frequently threaten the already compromised physical condition of the patients.

Ann Gen Psychiatry. 2007; 6: 25.

The patient was a 55-year-old married female, mother of two healthy grown-up children, with a history of type-1 DM since the age of 11. For her diabetes, she was initially treated with insulin (Novolente) once and then twice daily for 35 years. Subsequently, she was administered an intensified insulin regimen comprising insulin (NPH) twice daily and rapid-acting regular insulin three times daily pre-prandially, followed by a regimen of NPH and very-rapid acting insulin analogue (lispro) pre-prandially. During the last 3 years the patient has been treated with continuous subcutaneous insulin infusion through a pump, requiring 18–20 units/day as a basal regimen and a total of around 18–20 units/day as bolus injections. However, during the last year, due to the patient's poor compliance with the antidiabetic regimen and the required dietary pattern, her glucemic control deteriorated markedly, with frequent hypoglycemic episodes (and even comas) 2–3 times a week, followed by hyperglycemia events. In fact, the patient often felt hungry and, after the extra meals and fearing that her glucose level had risen excessively, she self-administered more insulin than required, thus causing the hypoglycemic episodes. Over the years she had also developed severe diabetic retinopathy, requiring laser treatments, as well as renal vascular complications of DM.

Ann Gen Psychiatry. 2007; 6: 25.

Despite the obvious severe limitations inherent to case reports in general, the present work suggests that OXC as monotherapy might be both safe and effective in the treatment of acute mania in patients with early-onset type-1 DM, whose already compromised physical condition constitutes an absolute or relative contra-indication to the administration of standard treatments.