Amelogenin-Derived Peptides in Bone Regeneration: A Systematic Review.

Amelogenins are enamel matrix proteins currently used to treat bone defects in periodontal surgery. Recent studies have highlighted the relevance of amelogenin-derived peptides, named LRAP, TRAP, SP, and C11, in bone tissue engineering. Interestingly, these peptides seem to maintain or even improve the biological activity of the full-length protein, which has received attention in the field of bone regeneration. In this article, the authors combined a systematic and a narrative review. The former is focused on the existing scientific evidence on LRAP, TRAP, SP, and C11's ability to induce the production of mineralized extracellular matrix, while the latter is concentrated on the structure and function of amelogenin and amelogenin-derived peptides. Overall, the collected data suggest that LRAP and SP are able to induce stromal stem cell differentiation towards osteoblastic phenotypes; specifically, SP seems to be more reliable in bone regenerative approaches due to its osteoinduction and the absence of immunogenicity. However, even if some evidence is convincing, the limited number of studies and the scarcity of in vivo studies force us to wait for further investigations before drawing a solid final statement on the real potential of amelogenin-derived peptides in bone tissue engineering.

Does Manual Drilling Improve the Healing of Bone-Hamstring Tendon Grafts in Anterior Cruciate Ligament Reconstruction? A Histological and Biomechanical Study in a Rabbit Model.

BACKGROUND: Heat necrosis due to motorized drilling during anterior cruciate ligament (ACL) reconstruction could be a factor in delayed healing at the bone-tendon graft interface. HYPOTHESIS: The process of osteointegration could be enhanced using manual drilling. It reduces the invasiveness of mechanical-thermal stress normally caused by the traditional motorized drill bit. STUDY DESIGN: Controlled laboratory study. METHODS: ACL reconstruction using semitendinosus tendon autografts was performed in 28 skeletally mature female New Zealand white rabbits, which were randomly divided into 3 groups. In group A (n = 12), the tunnels were drilled using a motorized device; in group B (n = 12), the tunnels were drilled using a manual drill bit; and group C (n = 4) served as a control with sham surgical procedures. The healing process in the tunnels was assessed histologically at 2, 4, 8, and 12 weeks and graded according to the Tendon-Bone Tunnel Healing (TBTH) scoring system. In addition, another 25 rabbits were used for biomechanical testing. The structural properties of the femur-ACL graft-tibia complex, from animals sacrificed at 8 weeks postoperatively, were determined using uniaxial tests. Stiffness (N/mm) and ultimate load to failure (N) were determined from the resulting load-elongation curves. RESULTS: The time course investigation showed that manual drilling (group B) had a higher TBTH score and improved mechanical behavior, reflecting better organized collagen fiber continuity at the bone-fibrous tissue interface, better integration between the graft and bone, and early mineralized chondrocyte-like tissue formation at all the time points analyzed with a maximum difference at 4 weeks (TBTH score: 5.4 [group A] vs 12.3 [group B]; P < .001). Stiffness (23.1 ± 8.2 vs 17.8 ± 6.3 N/mm, respectively) and ultimate load to failure (91.8 ± 60.4 vs 55.0 ± 18.0 N, respectively) were significantly enhanced in the specimens treated with manual drilling compared with motorized drilling (P < .05 for both). CONCLUSION: The use of manual drilling during ACL reconstruction resulted in better tendon-to-bone healing during the crucial early weeks. Manual drilling was able to improve the biological and mechanical properties of bone-hamstring tendon graft healing and was able to restore postoperative graft function more quickly. Tunnel drilling results in bone loss and deficient tendon-bone healing, and heat necrosis after tunnel enlargement may cause mechanical stress, contributing to a delay in healing. Manual drilling preserved the bone stock inside the tunnel, reduced heat necrosis, and offered a better microenvironment for faster healing at the interface. CLINICAL RELEVANCE: Based on study results, manual drilling could be used successfully in human ACL reconstruction, but further clinical studies are needed. A clinical alternative, called the original "all-inside" technique, has been developed for ACL reconstruction. In this technique, the femoral and tibial tunnels are manually drilled only halfway through the bone for graft fixation, reducing bone loss. Data from this study suggest that hamstring tendon-to-bone healing can be improved using a manual drilling technique to form femoral and tibial tunnels.

Biodegradable composite porous poly(dl-lactide-co-glycolide) scaffold supports mesenchymal stem cell differentiation and calcium phosphate deposition.

In recent decades, tissue engineering strategies have been proposed for the treatment of musculoskeletal diseases and bone fractures to overcome the limitations of the traditional surgical approaches based on allografts and autografts. In this work we report the development of a composite porous poly(dl-lactide-co-glycolide) scaffold suitable for bone regeneration. Scaffolds were produced by thermal sintering of porous microparticles. Next, in order to improve cell adhesion to the scaffold and subsequent proliferation, the scaffolds were coated with the osteoconductive biopolymers chitosan and sodium alginate, in a process that exploited electrostatic interactions between the positively charged biopolymers and the negatively charged PLGA scaffold. The resulting scaffolds were characterized in terms of porosity, degradation rate, mechanical properties, biocompatibility and suitability for bone regeneration. They were found to have an overall porosity of ∼85% and a degradation half time of ∼2 weeks, considered suitable to support de novo bone matrix deposition from mesenchymal stem cells. Histology confirmed the ability of the scaffold to sustain adipose-derived mesenchymal stem cell adhesion, infiltration, proliferation and osteo-differentiation. Histological staining of calcium and microanalysis confirmed the presence of calcium phosphate in the scaffold sections.

ß-NGF and ß-NGF receptor upregulation in blood and synovial fluid in osteoarthritis.

Osteoarthritis (OA) of the knee is the most common form of non-traumatic joint disease. Previous studies have shown the involvement of ß-NGF and its receptors TrKA and p75NTR in OA-related pain, but their role in its pathogenesis is still unclear. The aim of our study was to investigate the amount of ß-NGF and the expression levels of its receptors on cells isolated from synovial fluid and blood from OA patients who had undergone total knee arthroplasty, in order to check any possible correlation with the disease staging. Our results show a progressive stage-related increase of ß-NGF and its receptors both in serum and synovial fluid. Furthermore, with respect to control subjects, OA patients show an increased amount of inflammatory monocytes along with an increased expression of ß-NGF, TrKA and p75NTR. In conclusion, our study suggests a stage-related modulation of ß-NGF and its receptors in the inflammatory process of OA.

Trans-crocetin improves amyloid-ß degradation in monocytes from Alzheimer's Disease patients.

Herbal medicines have been recently employed in research and clinical studies for the potential treatment of behavioral and psychological symptoms associated with Alzheimer's Disease (AD) and other types of dementia. The present study investigates the effect of trans-crocetin, an active constituent of Crocus sativus L., to restore in vitro the reduced ability of AD patients' monocytes to degrade amyloid-ß(1-42) (Aß42). CD14+ monocytes from 22 sporadic AD patients with moderate cognitive impairment were isolated; then, the role of trans-crocetin, purified from saffron extracts, was evaluated in terms of Aß42 degradation rate through flow cytometry, as well as expression of cathepsin B by Western blotting. We observed that low micromolar doses of trans-crocetin enhanced Aß42 degradation in AD monocytes through the upregulation of the lysosomal protease cathepsin B. CA074Me, a potent and selective cathepsin B inhibitor, counteracted such trans-crocetin-induced effect. These data suggest that the carotenoid trans-crocetin improves in vitro the clearance of Aß42 through the involvement of cathepsin B, and this could be of value in developing a new anti-amyloid strategy in AD.

A role for NGF and its receptors TrKA and p75NTR in the progression of COPD.

Nerve growth factor and its receptors, TrkA and p75NTR, are involved in inflammation and airways diseases, but their role in chronic obstructive pulmonary disease is still unclear and not well investigated. our data indicate the stage dependent variation of nerve growth factor and its receptors in chronic obstructive pulmonary disease progression. In fact, for the first time, this study evaluates the presence of nerve growth factor and its receptors in serum and in peripheral blood mononuclear cells of patients with different stages of chronic obstructive pulmonary disease compared to healthy subjects, non-smoker and current smoker. Serum monocyte chemoattractant protein-1, tumor necrosis factor-α, interleukin-10 and forced expiratory volume in 1 s were also analyzed. Compared to healthy subjects, chronic obstructive pulmonary disease patients presented a staging-dependent increase in serum nerve growth factor, negatively correlated to forced expiratory volume in 1 s and positively to monocyte chemoattractant Protein-1. The percentage of p75NTR+ peripheral blood mononuclear cells increased in early stages of chronic obstructive pulmonary disease (I-II), while TrKA+ peripheral blood mononuclear cells increased in late stages (III-IV). Our data demonstrate the involvement and modulation of nerve growth factor and its receptors in chronic obstructive pulmonary disease and in its staging.

Biomechanical behaviour of native and sutured bronchi: An in-vitro study.

BACKGROUND: Biomechanical behaviour evaluation of a suture is an important information for the surgeon to choose the best technique to perform. OBJECTIVE: To assess the biomechanical behavior of the native and mechanically sutured bronchi. METHODS: Ten bronchi were harvested from slaughtered pigs and then randomly separated in two groups, a control intact group and a sutured group where specimens were cut in half and sutured, to evaluate mechanical properties during a tensile test using a loading frame machine. In addition optoelectric motion tracking system was used to evaluate suture profile motion during the test. RESULTS: Significant differences (p < 0.05) were found between the two groups for the parameters investigated. The control group showed a higher maximal stress resistance and stiffness than the suture group, while elongation at rupture was increased in the sutured group. All the sutures broke in symmetric manner, as the mean of the side difference of the sutured specimens was 0.93 ± 0.80 mm at rupture. CONCLUSIONS: Biomechanical behaviour of native and sutured bronchi was evaluated, giving highly reproducible parameters regarding mechanical properties that may help clinicians and bioengineers to rationalize the choice for a particular suture material or suture technique, increasing surgical outcomes.

The impact of nitric oxide on calcium homeostasis in PE/CA-PJ15 cells.

OBJECTIVE: Nitric oxide (NO) production and Ca(2+) homeostasis are key determinants for the control of many cell functions. NO is known to be a mediator of Ca(2+) homeostasis in a highly complex and cell-specific manner and although Ca(2+) homeostasis has been explored in human oral cancer cells, the exact mechanisms are not completely understood. In this study we investigated the impact of exogenous NO on [Ca(2+)]c homeostasis in PE/CA-PJ15 cells. DESIGN: Cells were treated with S-nitrosocysteine as NO-donor and the determinations of cytosolic Ca(2+) concentrations were performed using FURA-2 AM. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin were used to challenge mitochondrial functionality, whereas thapsigargin (TG) and La(3+) were employed to perturb intracellular calcium levels. RESULTS: NO derived from S-nitrosocysteine (CySNO) induced a dose-dependent reduction of cytosolic calcium [Ca(2+)]c whereas oxy-haemoglobin (oxyHb) completely counteracted this effect. Subsequently, we assessed possible relationships between NO and cellular structures responsible for Ca(2+) homeostasis. We found that uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and oligomycin strongly reduced the effect of NO on [Ca(2+)]c. Moreover, we found that during this mitochondrial energetic deficit, the effect of NO on [Ca(2+)]c was also reduced in the presence of La(3+) or thapsigargin. CONCLUSIONS: NO induces a concentration-dependent [Ca(2+)]c reduction in PE/CA-PJ15 human oral cancer cells and potentiates mitochondrial Ca(2+) buffering in the presence of TG or La(3+). Further, we show that exogenous NO deregulates Ca(2+) homeostasis in PE/CA-PJ15 cells with fully energized mitochondria.

miR128 up-regulation correlates with impaired amyloid ß(1-42) degradation in monocytes from patients with sporadic Alzheimer's disease.

Alzheimer's disease (AD), the most common form of dementia in elderly individuals, is characterized by neurofibrillary tangles, extracellular amyloid-ß (Aß) plaques and neuroinflammation. New evidence has shown that the lysosomal system might be a crossroad in which etiological factors in AD pathogenesis converge. This study shows that several lysosomal enzymes, including Cathepsin B, D, S, ß-Galactosidase, α-Mannosidase, and ß-Hexosaminidase, were less expressed in monocytes and lymphocytes from patients with a clinical diagnosis of AD dementia compared with cells from healthy controls. In vitro experiments of gain and loss of function suggest that down-regulation is a direct consequence of miR-128 up-regulation found in AD-related cells. The present study also demonstrates that miR-128 inhibition in monocytes from AD patients improves Aß(1-42) degradation. These results could contribute to clarify the molecular mechanisms that affect the imbalanced Aß production/clearance involved in the pathogenesis of AD.

Protein encapsulation in biodegradable polymeric nanoparticles: morphology, fluorescence behaviour and stem cell uptake.

The synthesis and characterization of new biodegradable polymeric NPs loaded with bovine serum albumin marked with fluorescein isothiocyanate (FITC-BSA) is reported. The protein is encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) NPs by the double emulsion method with subsequent solvent evaporation. The NPs display a spherical shape with a narrow size distribution and no aggregation is observed after drying. Steady-state and time-resolved fluorescence measurements appear to be a sensitive method to investigate the protein environment on the nanometer-scale. Finally, FITC-BSA-loaded NPs are rapidly internalized in stem cells. Interestingly, 25% cells were slightly positive after 28 days.

Expression of cathepsins S and D signals a distinctive biochemical trait in CD34+ hematopoietic stem cells of relapsing-remitting multiple sclerosis patients.

BACKGROUND: The elucidation of mechanistic aspects of relapsing-remitting multiple sclerosis (RRMS) pathogenesis may offer valuable insights into diagnostic decisions and medical treatment. RESULTS: Two lysosomal proteases, cathepsins S and D (CatS and CatD), display an exclusive pattern of expression in CD34(+) hematopoietic stem cells (HSCs) from peripheral blood of acute MS (A-MS) patients (n = 20). While both enzymes normally exist as precursor forms in the HSCs of healthy individuals (n = 30), the same cells from A-MS patients consistently exhibit mature enzymes. Further, mature cathepsins are expressed at lower rates in stable MS subjects (S-MS, n = 15) and revert to precursor proteins after interferon-ß1a treatment (n = 5). Mature CatD and CatS were induced in HSCs of healthy donors that were either co-cultured with PBMCs of A-MS patients or exposed to their plasma, suggesting a functional involvement of soluble agents. Following HSC exposure to several cytokines known to be implicated in MS, and based on relative cytokine levels displayed in A-MS, S-MS and control individuals, we identified IL-16 as a specific cell signaling factor associated with cathepsin processing. CONCLUSIONS: These data point to an evident correlation between CatS and CatD expression and MS clinical stage, and define a biochemical trait in HSCs with functional, medical, and diagnostic relevance.

Nitric oxide depletion alters hematopoietic stem cell commitment toward immunogenic dendritic cells.

BACKGROUND: NO* is a key molecule involved in the regulation of cell survival, proliferation and differentiation in many cell types. In this study we investigated the contribution of NO* during the differentiation of human peripheral blood hemopoietic stem cells (CD34+HSCs) toward immunogenic dendritic cells (i-DCs). METHODS: We depleted autocrine NO* production, using NG-monomethyl-L-arginine monoacetate (L-NMMA) and paracrine NO', using oxy-hemoglobin (HbO2) as a NO* scavenger during in vitro differentiation of CD34+HSCs to i-DCs. We monitored the NO* level, cell proliferation, phenotype and differentiation potential. RESULTS: We found that the depletion of paracrine or autocrine NO* correlated with (I) an active proliferation state at the end of differentiation, when control cells were not proliferating; (II) a significant reduction in the expression levels of differentiative markers (CD1a and HLA-DR) with a parallel high expression of the CD34 marker (III) with a retrieved clonogenic ability compared to control cells. CONCLUSIONS: On the whole, our data indicate that the depletion of NO* during the commitment stage blocks CD34+HSC differentiation into i-DCs and maintains an undifferentiated, highly proliferating cell population, indicating/revealing a novel role for NO* in the commitment of CD34+HSCs into i-DCs. GENERAL SIGNIFICANCE: The essential finding of the present study is that NO*, produced in HSCs by NOS enzymes, may act as autocrine and paracrine effectors regulating the in vitro differentiation process of CD34+-HSCs toward i-DCs.

Tuning multi/pluri-potent stem cell fate by electrospun poly(L-lactic acid)-calcium-deficient hydroxyapatite nanocomposite mats.

In this study, we investigated whether multipotent (human-bone-marrow-derived mesenchymal stem cells [hBM-MSCs]) and pluripotent stem cells (murine-induced pluripotent stem cells [iPSCs] and murine embryonic stem cells [ESCs]) respond to nanocomposite fibrous mats of poly(L-lactic acid) (PLLA) loaded with 1 or 8 wt % of calcium-deficient nanohydroxyapatite (d-HAp). Remarkably, the dispersion of different amounts of d-HAp to PLLA produced a set of materials (PLLA/d-HAp) with similar architectures and tunable mechanical properties. After 3 weeks of culture in the absence of soluble osteogenic factors, we observed the expression of osteogenic markers, including the deposition of bone matrix proteins, in multi/pluripotent cells only grown on PLLA/d-HAp nanocomposites, whereas the osteogenic differentiation was absent on stem-cell-neat PLLA cultures. Interestingly, this phenomenon was confined only in hBM-MSCs, murine iPSCs, and ESCs grown on direct contact with the PLLA/d-HAp mats. Altogether, these results indicate that the osteogenic differentiation effect of these electrospun PLLA/d-HAp nanocomposites was independent of the stem cell type and highlight the direct interaction of stem cell-polymeric nanocomposite and the mechanical properties acquired by the PLLA/d-HAp nanocomposites as key steps for the differentiation process.

Knock-down of HEXA and HEXB genes correlate with the absence of the immunostimulatory function of HSC-derived dendritic cells.

In an attempt to investigate whether the genetic defect in the HEXA and HEXB genes (which causes the absence of the lysosomal ß-N-acetyl-hexosaminidase), are related to the wide inflammation in GM2 gangliosidoses (Tay-Sachs and Sandhoff disease), we have chosen the dendritic cells (DCs) as a study model. Using the RNA interference approach, we generated an in vitro model of HEXs knock-down immunogenic DCs (i-DCs) from CD34(+)-haemopoietic stem cells (CD34(+)-HSCs), thus mimicking the Tay-Sachs (HEXA-/-) and Sandhoff (HEXB-/-) cells. We showed that the absence of ß-N-acetyl-hexosaminidase activity does not alter the differentiation of i-DCs from HSCs, but it is critical for the activation of CD4(+)T cells because knock-down of HEXA or HEXB gene causes a loss of function of i-DCs. Notably, the silencing of the HEXA gene had a stronger immune inhibitory effect, thereby indicating a major involvement of ß-N-acetyl-hexosaminidase A isoenzyme within this mechanism.

Development of a New Tool for 3D Modeling for Regenerative Medicine.

The effectiveness of therapeutic treatment based on regenerative medicine for degenerative diseases (i.e., neurodegenerative or cardiac diseases) requires tools allowing the visualization and analysis of the three-dimensional (3D) distribution of target drugs within the tissue. Here, we present a new computational procedure able to overcome the limitations of visual analysis emerging by the examination of a molecular signal within images of serial tissue/organ sections by using the conventional techniques. Together with the 3D anatomical reconstitution of the tissue/organ, our framework allows the detection of signals of different origins (e.g., marked generic molecules, colorimetric, or fluorimetric substrates for enzymes; microRNA; recombinant protein). Remarkably, the application does not require the employment of specific tracking reagents for the imaging analysis. We report two different representative applications: the first shows the reconstruction of a 3D model of mouse brain with the analysis of the distribution of the ß-Galactosidase, the second shows the reconstruction of a 3D mouse heart with the measurement of the cardiac volume.

Lysosomal ß-galactosidase and ß-hexosaminidase activities correlate with clinical stages of dementia associated with Alzheimer's disease and type 2 diabetes mellitus.

Multiple epidemiological studies have shown that individuals affected by type-2 diabetes mellitus (T2DM) carry a 2-to-5-fold higher risk of developing Alzheimer's disease (AD) when compared to non-diabetic subjects. Thus, biochemical parameters that can be easily and routinely assessed for high-confidence evaluation of diabetic conditions leading to AD (AD-T2DM) are regarded as efficient tools aimed at early diagnosis and, in turn, timely AD treatment. In this regard, the activity of lysosomal glycohydrolases may of use, in light of the implication of these enzymes in early events that underlie AD pathology and an overt correlation, in diabetes, between altered metabolic homeostasis, abnormal glycohydrolase secretion in body fluids, and occurrence of diabetic complications. Based on marked up-regulation previously shown in a peripheral, cell-based model of AD, we selected ß-Galactosidase, ß-Hexosaminidase, and α-Mannosidase to discriminate T2DM from AD-T2DM subjects. A screen of 109, 114, and 116 patients with T2DM, AD and AD-T2DM, respectively, was performed by testing enzyme activities in both blood plasma and peripheral blood mononuclear cells. Compared to age-matched, healthy controls (n = 122), ß-Galactosidase and ß-Hexosaminidase activities markedly diverged across the three groups, whereas virtually unchanged values were observed for α-Mannosidase. In particular, plasma ß-Galactosidase and ß-Hexosaminidase levels were higher in patients with AD-T2DM compared to those with T2DM, suggesting different mechanisms leading to enzyme secretion. Statistical analyses based on ROC curves showed that both ß-Galactosidase and ß-Hexosaminidase activities, either intracellular or plasma-secreted, may be used to discriminate AD patients from controls and AD-T2DM from T2DM patients.

Coordinated involvement of cathepsins S, D and cystatin C in the commitment of hematopoietic stem cells to dendritic cells.

The identity of biochemical players which underpin the commitment of CD34(+) hematopoietic stem cells to immunogenic or tolerogenic dendritic cells is largely unknown. To explore this issue, we employed a previously established cell-based system amenable to shift dendritic cell differentiation from the immunogenic into the tolerogenic pathway upon supplementation with a conventional cytokine cocktail containing thrombopoietin (TPO) and IL-16. We show that stringent regulation of cathepsins S and D, two proteases involved in antigen presentation, is crucial to engage cell commitment to either route. In response to TPO+IL-16-dependent signaling, both cathepsins undergo earlier maturation and down-regulation. Additionally, cystatin C orchestrates cathepsin S expression through a tight but reversible interaction that, based on a screen of adult stem cells from disparate origins, CD14(+) cells, primary fibroblasts and the MCF7 cell line, appears unique to CD34(+) stem cells from peripheral and cord blood. As shown by CD4(+) T cell proliferation in mixed-lymphocyte reactions, cell commitment to either pathway is disrupted upon cathepsin knockdown by RNAi. Surprisingly, similar effects were also observed upon gene overexpression, which prompts atypically accelerated maturation of cathepsins S and D in cells of the immunogenic pathway, similar to the tolerogenic route. Furthermore, RNAi studies revealed that cystatin C is a proteolytic target of cathepsin D and has a direct, causal impact on cell differentiation. Together, these findings uncover a novel biochemical cluster that is subject to time-controlled and rigorously balanced expression to mediate specific stem cell commitment at the crossroads towards tolerance or immunity.

Mechanotransduction: tuning stem cells fate.

It is a general concern that the success of regenerative medicine-based applications is based on the ability to recapitulate the molecular events that allow stem cells to repair the damaged tissue/organ. To this end biomaterials are designed to display properties that, in a precise and physiological-like fashion, could drive stem cell fate both in vitro and in vivo. The rationale is that stem cells are highly sensitive to forces and that they may convert mechanical stimuli into a chemical response. In this review, we describe novelties on stem cells and biomaterials interactions with more focus on the implication of the mechanical stimulation named mechanotransduction.

Efficient siRNA delivery by the cationic liposome DOTAP in human hematopoietic stem cells differentiating into dendritic cells.

RNA interference technology is an ideal strategy to elucidate the mechanisms associated with human CD34(+) hematopoietic stem cell differentiation into dendritic cells. Simple manipulations in vitro can unequivocally yield alloreactive or tolerogenic populations, suggesting key implications of biochemical players that might emerge as therapeutic targets for cancer or graft-versus-host disease. To knockdown proteins typically involved in the biology of dendritic cells, we employed an siRNA delivery system based on the cationic liposome DOTAP as the carrier. Freshly-isolated CD34(+) cells were transfected with siRNA for cathepsin S with negligible cytotoxicity and transfection rates (>60%) comparable to the efficiency shown by lentiviral vectors. Further, cathepsin S knockdown was performed during both cell commitment and through the entire 14-day differentiation process with repeated transfection rounds that had no effect per se on cell development. Tested in parallel, other commercially-available chemical reagents failed to meet acceptable standards. In addition to safe and practical handling, a direct advantage of DOTAP over viral-mediated techniques is that transient silencing effects can be dynamically appraised through the recovery of targeted proteins. Thus, our findings identify DOTAP as an excellent reagent for gene silencing in resting and differentiating CD34(+) cells, suggesting a potential for applications in related preclinical models.