Dental pulp stem cells (DPSC) have drawn much interest for the regeneration of mineralized tissues, and several studies have compared DPSC to bone marrow-derived mesenchymal stem cells (BMMSC). However, conflicting results, possibly due to donor-associated variability, have been published and the regenerative potential of DPSC is currently unclear. In the present study we have sought to address this problem using a donor-matched experimental design to robustly compare the biological properties of DPSC and BMMSC. All experiments were performed using cells isolated from a single adult Sprague-Dawley rat. Our results show that DPSC and BMMSC had similar morphologies and flow cytometry profiles, were capable of forming colonies in vitro, and were capable of osteogenic, chondrogenic, and adipogenic differentiation. However, quantitative comparisons revealed that DPSC had a faster population doubling time and a higher percentage of stem/progenitor cells in the population as determined by clonogenic assays. Furthermore, while both cell populations formed mineral in vitro, DPSC had significantly higher alkaline phosphatase activity than BMMSC after three weeks in osteogenic medium. These data show several key differences between DPSC and BMMSC and support the possibility of using DPSC for mineralized tissue regeneration.

Objective-Standard competitive repopulation assays have proven valuable in evaluating engraftment potential in ablated hosts, permitting comparisons between various test cell populations. However, no similar method exists to compare engraftment of test cells in submyeloablated hosts, which would be helpful given the applications of reduced-intensity conditioning for hematopoietic gene-replacement therapy and other cellular therapies. Here, we developed a novel assay to quantitate engraftment of hematopoietic stem cells in submyeloablated hosts.Methods-Engraftment of murine marrow cells transduced with retroviral vectors using two separate protocols was compared to engraftment of fresh untreated competitor cells within low-dose radiation conditioned hosts using a "three-way" marking system, so that test, competitor and host cell chimerism could be reliably determined post-transplant.Results-We demonstrate that the repopulating ability of marrow cells transduced using two distinct protocols was reduced ~10-fold compared to fresh competitor cells in submyeloablated hosts utilizing the novel "three-way" transplant assay.Conclusions-Murine marrow cells transduced using a clinically-applicable protocol acquire an engraftment defect in submyeloablated hosts, similar to cells transduced using a research protocol. We conclude that the submyeloablative competitive repopulation assay described here will be of benefit to comparatively assess the engraftment ability of manipulated hematopoietic stem cells using various culture protocols, such as to test the impact of modifications in transduction protocols needed to attain therapeutic levels of gene-corrected blood cells, or the effect of ex vivo expansion protocols on engraftment potential.

Objective We recently reported that murine marrow cultured ex vivo for gamma-retrovirus transduction engrafts ~10 fold less well than fresh marrow upon transplantation into submyeloablated hosts. Here, we evaluated homing efficiency as a potential mechanism for this engraftment disparity, and whether CD26 inhibition with the tripeptide Diprotin A (DipA) would enhance engraftment of ex vivo cultured cells in submyeloablated hosts. Methods Homing and engraftment of fresh and ex vivo cultured lineage-negative (lin-) marrow cells in submyeloablated congenic hosts with and without DipA treatment was evaluated. Expression of CXCR4 and CD26 on fresh and cultured lin- marrow cells was compared. Results Homing of lin- cells cultured for gamma-retrovirus transduction was ≥3-fold less than that of fresh lin- cells 20 hours after transplantation into submyeloablated hosts. DipA treatment of fresh lin- cells resulted in ≥-fold increased homing and engraftment in submyeloablated hosts. DipA treatment, however, did not significantly improve homing or engraftment of cells undergoing a three-day culture protocol for gamma-retrovirus transduction in submyeloablated hosts. CXCR4 expression on lin- cells was significantly decreased following three days of culture; CXCR4 expression was not significantly altered following overnight culture. Conclusions Ex vivo culture of lin- cells for gamma-retroviral transduction downregulates CXCR4 expression and markedly impairs homing and engraftment of murine lin- marrow in submyeloablated hosts. While inhibition of CD26 activity with DipA increases homing and engraftment of fresh lin- cells, DipA treatment does not improve homing and engraftment of cultured lin- marrow cells in submyeloablated congenic hosts.

Background: There is an uncertainty and paucity of data regarding normal levels of disaccharidase activities (DA) in infants. Aim: To establish normal values for DA in infants. To determine the relationship between symptoms, intestinal mucosal histology and DA. Methods: Histology and DA of intestinal mucosal specimens from 131 infants (75 males; mean age 180 d; age range, 20-364 d) obtained endoscopically over an 8 year period were reviewed. Patients were divided into 2 groups based on absence (Group 1; nϭ63) or presence (Group 2; nϭ68) of failure to thrive (FTT) and/or diarrhea. These groups were further subdivided into 3 subgroups based on histological findings: (normal: A, mild abnormalities: B, and moderate/severe changes: C). Results: DA from group 1A represent normal values as these infants were free of FTT/diarrhea, and had normal intestinal mucosal histology (Table). Differences in DA were not dependent on symptoms, in absence of histological abnormalities (groups 1A and 2A), but rather on presence of histological changes even in the absence of symptoms (groups 1A vs 1B). Also, differences were found when patients with FTT and/or diarrhea with abnormal histology were compared to patients with no FTT and/or diarrhea with a normal brush border (groups 2B and 2C vs 1A). Conclusions: We describe normal levels of DA in infants. These may serve as reference values for clinical practice and laboratory analyses. Additionally, DA were related to mucosal histology and not certain symptoms. GROWTH OF INFANTS WITH BRONCHOPULMONARY DSPLASIA AFTER DISCHARGE.J Hesser, C Siegel, M Weiss, C Sajous. Loyola University Medical Center (LUMC), Maywood, IL.Purpose of Study: The objective of our study is to compare the pre-and post-discharge growth of infants with severe BPD enrolled in the LUMC Neonatal Home Care program to the growth after discharge of other infants with mild BPD seen at the neonatal follow-up clinic.Design and Methods: Adequate growth velocity of premature infants is 26 to 40 grams per day at one month corrected gestational age (GA). In this prospective study, 70 infants admitted to LUMC NICU from 9/1/04 until 10/31/05 with a birth weight Յ 1500 grams were enrolled. Infants with an O2 requirement for Ն 28 days were selected. LUMC Home Care Program follows infants discharged home requiring O2, NG feedings, monitors or special needs. Infants requiring O2 at home were the severe BPD group, and infants without home care and on room air were the mild BPD group. While in the NICU, length, weight and head circumference were recorded weekly in nutrition rounds. After discharge, the severe BPD group had their weight and length recorded at each home visit up to 6 months corrected GA, according to the severity of their disease. The mild BPD group had data collected at the NICU Follow-Up Clinic. Feedings and average daily calories were recorded.Results: 70 patients completed the study. 39 were in the severe BPD group, and 31 were in the mild group. The data was analyzed with the student t-test. Conclusion: While in the ...

We previously demonstrated that engraftment of murine whole bone marrow (WBM) transduced with an oncoretroviral vector using an optimized 5-fluorouracil (5-FU)-based transduction protocol is reduced ~3-fold compared to fresh WBM upon transplantation into sublethally irradiated hosts, although competitive repopulating ability in ablated hosts is not decreased (Goebel et al., Exp. Hematol. 30:1324, 2002). We therefore sought to determine whether marrow cells transduced using a clinically relevant, non-5-FU-containing protocol would engraft more efficiently. Li et al. (Exp. Hematol. 31:1206, 2003) showed that lineage-depleted (lin−) marrow cells from donor mice not treated with 5-FU were effectively transduced, and repopulated myeloablated hosts. We hypothesized that ex vivo culture for gene transfer in the absence of 5-FU would lead to improved donor marrow engraftment in submyeloablated hosts. Lin− cells, isolated from B6.SJL (Boy J; CD45.1+) WBM using a Miltenyi kit and VarioMACS apparatus, were prestimulated in StemSpan serum-free medium with SCF and IL-6 for 48 hours, followed by overnight transduction on RetroNectin-coated plates preloaded with ecotropic SF1-EGFP retroviral supernatant. Cell recovery from the MACS column (1.9 ± 1.4%), bulk transduction efficiency (77.3 ± 12%), and lin− cell purity (58 ± 17%; all from 7–13 experiments) was similar to that previously described. Transplantation of 106 lin− transduced cells into 300 cGy-conditioned congenic C57Bl6/J (B6; CD45.2+) hosts produced only 1.4 ± 0.5% donor chimerism 4–6 months post-transplant, significantly lower than that observed using 106 fresh lin− cells (29 + 18.8%; N = 8–9 hosts each from 2–3 experiments). The percentage of EGFP+ cells in the donor population, nevertheless, was 55.6 ± 18%, indicating that stem cells were marked but engrafted poorly. The repopulating ability of transduced lin− marrow was reduced ~10-fold compared to fresh lin− cells as determined in competitive repopulation assays in ablated hosts. Together, these data suggest that lin cells cultured ex vivo for gene transfer acquired an engraftment defect despite the absence of 5-FU. Increasing the conditioning radiation dose to 550 cGy, a dose used in prior canine and non-human primate gene transfer studies, markedly improved donor chimerism following transplantation of 106 fresh lin− cells (90 ± 1.3% at 4 months, N = 5) or 106 transduced lin− cells (38.5 ± 14% at 2 months, N = 10), suggesting that greater reduction in host stem cell function may be needed for engraftment of cells cultured ex vivo for gene transfer. Ongoing studies to investigate the mechanism responsible for this engraftment defect indicate that expression of adhesion molecules important for homing and engraftment (CD29, 44, 49d, 49e, 62L), CXCR4 expression, and the percentage of cells actively cycling are not significantly altered by the transduction process, although functional studies are underway. The percentage of Sca-1+lin−c-kit+ (SLK) cells in the transduced cell pool is similar to that of freshly isolated lin− cells; thus, transplantation of lin− cells cultured ex vivo for gene transfer results in significantly lower donor chimerism than fresh lin− cells despite the grafts containing similar numbers of SLK cells. Secondary transplants and limiting dilution studies to determine stem cell self-renewal and engraftment capacity before and after ex vivo culture for gene transfer are in progress.