Regulation of Biotin Transport in Saccharomyces cerevisiae

The metabolic control of biotin transport in Saccharomyces cerevisiae was investigated. Nonproliferating cells harvested from cultures grown in excess biotin (25 ng/ml) took up small amounts of biotin, whereas cells grown in biotin-sufficient medium (0.25 ng/ml) accumulated large amounts of the vitamin. Transport was inhibited maximally in cells grown in medium containing 9 ng (or more) of biotin per ml. When avidin was added to biotin-excess cultures, the cells developed the ability to take up large amounts of biotin. Boiled avidin was without effect, as was treatment of cells with avidin in buffer. Avidin did not relieve transport inhibition when added to biotin-excess cultures treated with cycloheximide, suggesting that protein synthesis was required for cells to develop the capacity to take up biotin after removal of extracellular vitamin by avidin. Cycloheximide did not inhibit the activity of the preformed transport system in biotin-sufficient cells. The presence of high intracellular free biotin pools did not inhibit the activity of the transport system. The characteristics of transport in biotin-excess cells (absence of temperature or pH dependence, no stimulation by glucose, absence of iodoacetate inhibition, independence of uptake on cell concentration, and nonsaturation kinetics) indicated that biotin entered these cells by diffusion. The results suggest that the synthesis of the biotin transport system in S. cerevisiae may be repressed during growth in medium containing high concentrations of biotin.     

Lymphocyte propionyl-CoA carboxylase and accumulation of odd-chain fatty acid in plasma and erythrocytes are useful indicators of marginal biotin deficiency☆

Background: Recent studies indicate marginal biotin deficiency is more common than previously thought. That conclusion’s validity rests on two indicators of biotin status that depend on renal function.Objective: Assessing the validity of two indicators of biotin status that do not depend upon renal function: 1) activity of the biotin-dependent enzyme propionyl-CoA carboxylase (PCC) in lymphocytes and 2) accumulation of odd-chain fatty acids in the lipids of plasma and erythrocytes.Design: Marginal biotin deficiency was induced in 11 healthy adults by egg-white feeding for 28 days. Blood and 24-h urine samples were collected before commencing the diet and twice weekly thereafter. After depletion, biotin status was restored with a general diet with or without 80 μg/day or 328 nmol/day biotin supplement. Activity of PCC was determined by an optimized NaH ¹⁴CO₃ incorporation assay. Fatty acid composition was determined by gas chromatography.Results: With time on the egg-white diet, lymphocyte PCC activity decreased significantly (P <0.0001); C15:0 and C17:0 content increased significantly in the lipids of plasma and erythrocytes (P <0.015). In eight of 11 subjects, lymphocyte PCC activity returned to normal within three weeks of resuming general diets with or without biotin supplement. With repletion, C15:0 and C17:0 in plasma lipids decreased (P <0.02), but odd-chain content of erythrocytes did not decrease significantly.Conclusions: Lymphocyte PCC activity is an early and sensitive indicator of marginal biotin deficiency. Odd-chain fatty acids accumulate in blood lipids more gradually during marginal deficiency and return to normal more gradually after biotin repletion.     

Immunoregulatory activity of culture-induced suppressor macrophages

Rat splenic cells precultured in vitro for 5 days exhibited marked suppressive activity on the secondary cytotoxic T lymphocyte (CTL) response to a Gross virus-induced lymphoma. Suppressive activity was produced by macrophages (MØ) rather than lymphocytes and as low as 1% MØ content was sufficient to achieve completely inhibited CTL responses. Aspirin, indomethacin, and d,l-6-chloro-2-methylcarbazole-2-acetic acid prevented cultured splenic MØ from exerting their inhibitory effect, thereby suggesting a role for prostaglandins in suppression. Events which occurred within the first 24 to 48 hr of the CTL response were susceptible to the suppressive action of MØ since normal CTL responses were obtained if suppressive MØ were added later than Day 2 or if indomethacin was added within the first 24 to 48 hr of culture. Two processes of lymphocyte activation, namely blast transformation and DNA synthesis, were inhibited in the presence of suppressive MØ. However, suppression of these processes did not result in the loss of CTL progenitor cells since CTL responses that were inhibited in the presence of suppressive MØ proceeded normally following their removal.     

Lyt-2+ suppressor T cells are resistant to anti-Lyt-2 antibody blocking

Lyt-2 molecules play a role in antigen recognition by cytotoxic T lymphocytes (CTL). In an attempt to determine whether Lyt-2 molecules play a similar role in suppressor T cell (Ts) functions, the effect of anti-Lyt-2 antibodies on Ts generation and effector activity was studied. Allospecific Ts were induced in allogeneic mixed lymphocyte cultures (MLC). Anti-Lyt-2 antibodies added to MLC in the absence of complement abolished CTL generation, but had no effect on concomitant induction of Ts. In a different experimental system, allospecific Ts were induced in cultures treated with pyrilamine, which blocks generation of CTL but allows differentiation of Ts. The addition of anti-Lyt-2 antibodies to pyrilamine-treated MLC resulted in unaffected induction of Ts. It was further demonstrated that the effector activity of Ts was as resistant to anti-Lyt-2 antibodies as their induction, in contrast to the cytolytic activity of CTL, which was inhibited by the same antibodies. Ts in the present experimental system were Lyt-2+ antigen-specific cells. It therefore appears that Lyt-2 molecules, although expressed on both CTL and Ts, are involved in CTL activity, but do not play an essential role in Ts function.     

Enhanced neogenic potential of Panc-1 cells supplemented with human umbilical cord blood serum--An alternative to FCS

The promise(s) of using Fetal Calf Serum (FCS) as a supplement for the maintenance of cell cultures has been well documented. However, FCS forms the xenogenic source for any human derived cells/organ and limits its application. Recently, the usage of human umbilical cord blood serum (hUCBS) for maintenance of mesenchymal cells has been supportive. In the present study we investigated the effects of hUCBS and FCS on the proliferation (viability, proliferative) and its differentiation potential (DTZ staining, immunofluroscence) to generate islet like cellular aggregates (ICAs) using the human derived Panc-1 cell lines. A comparative analysis of hUCBS and FCS for each parameter demonstrated that hUCBS supplemented media was better for proliferation and differentiation of the Panc-1 cells. The ICAs obtained from hUCBS primed cultures showed a higher yield, increased islet size, and showed an increase for insulin staining compared to FCS. We suggest that hUCBS can be explored as an alternate serum supplement for FCS, making it more feasible in cell systems of human derived origin and can also find its application for the human transplantation programmes.     

Activation and mechanism of action of suppressor macrophages

Intravenous administration of Corynebacterium parvum to alloimmunized mice activates splenic suppressor macrophages that effectively curtail primary and secondary generation of cytotoxic T lymphocytes (CTLs) in vitro. CTL generation was significantly inhibited in suppressed primary cultures by Day 3, the earliest time point that activity is first detected in control cultures. Suppressor macrophages had to be present during the first 24–48 hr of culture to effectively curtail the generation of CTLs. However, if suppressor macrophages were reactivated by 48-hr in vitro culture and then added to primary sensitizations that had been initiated 48 hr previously, they were capable of significant suppression. Suppressor cells produced a soluble factor that mediated the inhibition of CTL generation. The production or action of this factor could not be counteracted by indomethacin.     

Transforming growth factor-beta inhibits the in vitro generation of lymphokine-activated killer cells and cytotoxic T cells

Summary The effect(s) of purified transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) on the induction and function of lymphokine-activated killer (LAK) cells and cytotoxic T lymphocytes (CTL) was examined. The addition of TGF-beta, but not PDGF, to cultures containing fresh C57BL/6 mouse splenocytes or human peripheral blood lymphocytes plus recombinant interleukin-2 markedly inhibited the development of mouse and human LAK cell activity (measured after 3 days for cytotoxicity against cultured or fresh tumor targets in 4-h ⁵¹Cr release assays). The addition of TGF-beta, but not PDGF, to a one-way, C57BL/6 anti-DBA/2, mixed lymphocyte reaction effectively blocked the generation of allospecific CTL as well. However, TGF-beta did not inhibit the effector function of LAK cells or of allospecific CTL when added directly to the short-term cytolytic assay. A second form of homodimeric TGF-beta, type 2, was also found to be suppressive on the development of murine LAK cells and allospecific CTL. Collectively, these data demonstrate that the peptide TGF-beta is a potent inhibitor of LAK cell and CTL generation in vitro.     

Production of T cell differentiation factor in syngeneic lymphocyte macrophage culture for cytotoxic T lymphocyte generation

This study demonstrated that T cell differentiation factor (TCDF) was produced in syngeneic lymphocyte-macrophage cultures. Conditioned medium containing TCDF and interleukin 2 (IL 2) induced the differentiation of leukoagglutinin (LA)-activated cytotoxic T lymphocyte precursors (CTLp) into cytotoxic T lymphocyte (CTL) effectors. The production of TCDF and IL 2 peaked at day 4 to 5 in cultures containing normal spleen cells, syngeneic peritoneal macrophages, and indomethacin. Macrophages and T cells with Thy-1+, L3T4+, and Lyt-2- phenotype were needed for TCDF production. There was no requirement for xenogeneic serum in the culture medium; thus, TCDF could be produced in a syngeneic system. Recognition of self Ia molecules appeared to be essential for TCDF production, which was completely abolished by the addition of monoclonal anti-Ia antibody. In our experiments, removal of IL 2 from conditioned medium containing TCDF abolished its ability to generate LA-activated CTL. However, the cytotoxic response could be restored by the addition of a small amount (5 U/ml) of purified human recombinant IL 2 (HRIL 2), which alone was unable to generate LA-activated CTL at this dose. The generation of LA-activated CTL by high dose HRIL 2 (greater than 50 U/ml) was likely due to the endogenous production of TCDF. The bulk of TCDF could be separated from IL 2 by gel filtration in a Sephadex G-100 column. The peak of TCDF activity was concentrated at a m.w. of 16K dalton, and there was very little IL 2 activity in these fractions. When added alone to the LA-activated lymphocyte cultures, these active fractions were unable to induce CTL; supplementation of exogenous IL 2 was needed to restore the cytotoxic responses. Our findings indicate that both IL 2 and TCDF, which are needed in CTL generation. are produced in syngeneic cultures in the absence of antigenic or mitogenic stimulation.     

Genetic analysis and characterization of a Caulobacter crescentus mutant defective in membrane biogenesis.

A mutant of Caulobacter crescentus has been isolated which has an auxotrophic requirement for unsaturated fatty acids or biotin for growth on medium containing glucose as the carbon source. This mutant exhibits a pleiotropic phenotype which includes (i) the auxotrophic requirement, (ii) cell death in cultures attempting to grow on glucose in the absence of fatty acids or biotin, and (iii) a major change in the outer membrane protein composition before cell death. This genetic lesion did not appear to affect directly a fatty acid biosynthetic reaction because fatty acid and phospholipid syntheses were found to continue in the absence of supplement. Oleic acid repressed fatty acid biosynthesis and induced fatty acid degradation in the wild-type parent, AE5000 . The mutant strain, AE6000 , was altered in both of these regulatory functions. The AE6000 mutant also showed specific inhibition of the synthesis of outer membrane and flagellar proteins. Total phospholipid, DNA, RNA, and protein syntheses were unaffected. The multiple phenotypes of the AE6000 mutant were found to cosegregate and to map between hclA and lacA on the C. crescentus chromosome. The defect in this mutant appears to be associated with a regulatory function in membrane biogenesis and provides evidence for a direct coordination of membrane protein synthesis and lipid metabolism in C. crescentus.     

Beta (1-3) glucans as a probe for T cell specific immune adjuvants. II. Enhanced in vitro generation of cytotoxic T lymphocytes.

The capacity of five antitumor polysaccharides (PS) of the β (1–3) glucan type, lentinan, pachyman, pachymaran, carboxymethylpachymaran and hydroxyethylpachyman to augment the in vitro generation of alloreactive cytotoxic T lymphocytes (CTL) in a primary murine mixed lymphocyte culture was tested in detail. All of the PS tested, each at its own optimal concentration, induced enhanced alloreactive CTL responses. Lentinan, pachymaran and hydroxyethylpachyman increased the magnitude of the in vitro CTL responses up to 28 fold. The PS tested appeared to enhance the responsiveness of alloreactive prekiller T cells rather than the immunogenicity of the stimulator cells. Kinetic experiments indicated that in vitro CTL responses could be augmented when PS were added as late as 72 hr after initiation of the cultures, suggesting that PS influences the differentiation of antigenically triggered CTL precursors into cytotoxic effector cells. PS also augmented in vitro the generation of H-2 restricted hapten-specific CTL. The results indicate that the PS tested are effective immune adjuvants of both alloreactive CTL responses and H-2 restricted CTL responses specific to foreign antigens.     

Growth stimulation of rous sarcoma virus-transformed BHK cells by biotin and serum lipids

Biotin or a serum lipid extract stimulated proliferation of G1 arrested Rous sarcoma virus-transformed BHK cells in modified Eagle's MEM (BM). The cells could be maintained continuously in BM plus biotin (BMB), but not in BM plus serum lipid extract (BM X L). Avidin inhibited growth stimulation when added to BMB, but did not inhibit growth when added to BM X L. 14C-acetate incorporation into total cellular lipids was stimulated in BMB, but not in BM. Thin-layer chromatography of the labeled cellular lipid extract indicated that relatively large amounts of 14C-acetate were incorporated into phosphatidylserine and little into the other major phospholipids. In the neutral lipids, the largest amount of incorporation was in cholesterol. G1 arrested cells multiplied rapidly in BM supplemented with dialyzed serum (BM X DS), but they did not multiply in BM with delipidized serum (BM X DLS). The addition of biotin or serum lipid extract to BM X DLS stimulated growth. Growth stimulation in BM X DLS by biotin was inhibited by avidin, but avidin had no effect on growth stimulation by serum lipid extract. Biotin stimulated additional multiplication in BM X DS and avidin inhibited this additional growth stimulation. These results suggest that growth stimulation requires lipids supplied by serum lipids or by de novo synthesis stimulated by biotin. In the absence of serum, the stimulation of the synthesis of growth factor(s) by biotin are also required for continuous multiplication.     

Interruption of the adipose conversion of 3T3 cells by biotin deficiency: differentiation without triglyceride accumulation

During the adipose conversion of 3T3 cells in medium supplemented with 10% serum, the accumulation of triglyceride depends upon a small molecular weight component in the serum. When this is removed by exhaustive dialysis of the serum, the cells undergo some changes that are part of the adipose conversion, but very little triglyceride accumulates. After the addition of either a serum dialysate or commercial biotin, cellular lipid begins to accumulate rapidly. The dialyzable factor in the serum has numerous chemical properties of biotin.When cells begin the adipose conversion in the absence of biotin, they are unable to increase their rate of acetate incorporation into triglyceride, but they do undergo the same change in shape to spherical, the same increase in activity of cytoplasmic (NAD-linked) glycerophosphate dehydrogenase (300 fold) and the same increase in lipoprotein lipase (>50 fold) as in the presence of biotin. On the other hand, in the biotin-deficient state the activity of glycerophosphate acyltransferase and of malic enzyme rises to only a small fraction of the control level. After the addition of biotin to the biotin-deficient cultures, the activity of both enzymes increases to the control level within 24–48 hr.These results are consistent with the concept that the glycerophosphate dehydrogenase is a primary enzyme in the adipose conversion: its response (measured as the amount of extractable activity) does not depend upon the rate of cellular fatty acid synthesis. Lipoprotein lipase is similarly independent. On the other hand, both malic enzyme and glycerophosphate acyltransferase may be considered as secondary enzymes in the sense that their response during the adipose conversion is linked to the supply of fatty acid.     

Effect of fetal and adult bovine albumins on human lymphocyte proliferation in vitro

Fetal and adult bovine albumins were isolated and compared with the commercially obtained fraction V of Cohn, in terms of their ability to support cell proliferation, when added as a supplement to serum-free lymphocyte cultures activated with phytohemagglutinin. It was found that fetal albumin is the least efficient of the three proteins in permitting lymphoproliferation. A difference between the kinetics of proliferation of lymphocyte cultures supplemented with albumin and with serum was also observed.     

Inhibition of the lytic phase of murine T-cell-mediated alloimmune cytotoxicity by a rat antiactivated T-cell antiserum

Antisera produced in rats by immunization with alloimmune murine C57Bl/6 anti-P815 splenic lymphocytes or purified T cells activated in vitro by coculture with phytohemagglutinincoated L-929 cells were found to inhibit the in vitro cytolytic action of in vivo and in vitro alloimmune C57Bl/6 anti-P815 cytotoxic T cells in a 4-hr chromium-51 release assay. The rat anti-murine-activated lymphocyte (anti-MAL) or antiactivated T-cell (anti-ATC) serum inhibited lysis in the absence of exogenously added complement activity and were not directly cytotoxic to CTL. Absorption of anti-MAL with target cells P815, L-929, EL-4, and normal C57Bl/6 lymphocytes removed a limited amount of the CTL-inhibitory activity. In contrast, lectin-activated alloimmune lymphocytes fully absorbed the inhibitory activity indicating these antisera preferentially recognize unique antigenic determinants associated with the activated CTL cell surface. The anti-ATC was found to block alloimmune lysis by CTL from several inbred mouse strains suggesting these antisera recognized antigenic determinants of a common lytic mechanism. A kinetic analysis of the inhibitory activity of the anti-MAL on the CTL reaction scheme revealed this antiserum inhibited lysis at a post-Ca²⁺-dependent step, presumably during the target cell lytic phase. This result suggests the rat antiserum can neutralize the CTL lytic mechanism.     

Inhibition of cytotoxic T lymphocyte-mediated lysis by ETYA: effect independent of arachidonic acid metabolism

Cytotoxic T lymphocyte (CTL)-mediated lysis of target cells was inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA) and other inhibitors of the lipoxygenase pathway at concentrations that inhibited arachidonic acid metabolism in mixed lymphocyte cultures. Inhibition was reversible and selective for the "lethal hit" stage in the CTL-target interaction. Studies to define CTL-specific arachidonic acid metabolites demonstrated that cloned CTL populations have little or no capacity to metabolize arachidonic acid. Therefore, inhibitor actions appear to be independent of the effects on CTL arachidonic acid metabolism. Alternative explanations for inhibitory effects are discussed.     

The role of IL-2 and T4+ cells in the generation of human influenza virus-specific CTL activity

Stimulation of human peripheral blood lymphocytes (PBL) with influenza A virus leads to the generation of virus-specific cytotoxic T lymphocyte (CTL) activity as well as natural killer (NK)-like activity. In this study, we show that exogenous IL-2 augments the in vitro generation of virus-specific CTL activity, only when added some days after the initiation of the culture. Apparently, the endogenously produced IL-2 can be a limiting factor in the in vitro generation of CTL activity. The increase of influenza virus-specific CTL activity after addition of exogenous IL-2 does not affect the restriction pattern of the CTL response. So, the preferential use of certain HLA antigens as restriction elements is not due to a limiting amount of endogenously produced IL-2. Depletion of T4+ cells completely abrogates the generation of virus-specific CTL activity. Addition of exogenous IL-2 to T4+-cell-depleted cultures fully restores the generation of HLA-restricted virus-specific CTL activity. We conclude that in the in vitro generation of virus-specific CTL activity in bulk cultures of human PBL the sole function of T4+ cells in human virus-specific CTL generation is the production of IL-2, no cognitive cell interaction of T8+ CTL precursors with T4+ cells is required, and in bulk cultures T8+ cells themselves are not able to produce sufficient amounts of IL-2 to ascertain the maturation of virus-specific CTL precursors into cytolytic T cells. Finally, we show that exogenous IL-2 also has a stimulatory effect on the NK-like or lymphokine-activated killer activity, which is always concomitantly induced in virus-specific CTL generation cultures, but has no influence on the levels of IFN produced in such cultures.     

Effect of serum concentration on the cytotoxicity of clay particles

Nanoparticle cytotoxicity testing based on in vitro methods frequently lack consistency. Even the inclusion of the commonly employed growth supplement, FCS (fetal calf serum), generates variable results. Thus, our object was to investigate the effect of FCS concentration on the cytotoxic behaviour of the unmodified nanoclay, Cloisite® Na⁺. Human monocytic U937 cells in medium supplemented with 5% FCS, 2.5% FCS or serum‐free medium were treated with 1 mg/ml Cloisite Na⁺. Cell growth in 2.5% FCS was significantly inhibited by Cloisite Na⁺ within 48 h, whereas little effect was seen with a supplement of 5% FCS. Without serum, cell growth was inhibited and Cloisite Na⁺ had a detrimental effect on these cells. In media supplemented with FCS, the nanoclays agglomerated together to form large bundles, whereas they were evenly dispersed throughout the medium in the absence of serum. Clay particles, therefore, have cytotoxic properties that may be linked to their dispersion pattern. These adverse effects seem to be masked by 5% FCS. Serum supplementation is an important consideration in the toxicological assessments of nanomaterials on cells, which needs to be addressed in the standardization of in vitro testing methods.     

Lymphocyte propionyl-CoA carboxylase and accumulation of odd-chain fatty acid in plasma and erythrocytes are useful indicators of marginal biotin deficiency small star,filled

BACKGROUND: Recent studies indicate marginal biotin deficiency is more common than previously thought. That conclusion's validity rests on two indicators of biotin status that depend on renal function.OBJECTIVE: Assessing the validity of two indicators of biotin status that do not depend upon renal function: 1) activity of the biotin-dependent enzyme propionyl-CoA carboxylase (PCC) in lymphocytes and 2) accumulation of odd-chain fatty acids in the lipids of plasma and erythrocytes.DESIGN: Marginal biotin deficiency was induced in 11 healthy adults by egg-white feeding for 28 days. Blood and 24-h urine samples were collected before commencing the diet and twice weekly thereafter. After depletion, biotin status was restored with a general diet with or without 80 &mgr;g/day or 328 nmol/day biotin supplement. Activity of PCC was determined by an optimized NaH 14CO(3) incorporation assay. Fatty acid composition was determined by gas chromatography.RESULTS: With time on the egg-white diet, lymphocyte PCC activity decreased significantly (P <0.0001); C15:0 and C17:0 content increased significantly in the lipids of plasma and erythrocytes (P <0.015). In eight of 11 subjects, lymphocyte PCC activity returned to normal within three weeks of resuming general diets with or without biotin supplement. With repletion, C15:0 and C17:0 in plasma lipids decreased (P <0.02), but odd-chain content of erythrocytes did not decrease significantly.CONCLUSIONS: Lymphocyte PCC activity is an early and sensitive indicator of marginal biotin deficiency. Odd-chain fatty acids accumulate in blood lipids more gradually during marginal deficiency and return to normal more gradually after biotin repletion.     

IL-4 (B cell-stimulatory factor 1) regulates multiple aspects of influenza virus-specific cell-mediated immunity

The effect of endogenous and exogenous IL-4 on the generation of influenza virus-specific cell-mediated immunity was examined. When added at the onset of the culture, IL-4 augmented both cluster of differentiation (CD)8+ lymphoproliferation and MHC-restricted, influenza virus-specific cytotoxicity. When added 5 or 6 days after initiation of cultures, IL-4 was highly effective at augmenting cytotoxicity, whereas no augmentation of proliferation was observed. This disassociation of the effect of IL-4 on lymphoproliferation and cytotoxicity indicated that IL-4 was providing a late signal in CTL generation. Studied at the level of CTL precursor maturation in microcultures, IL-4 was found not to increase cytotoxicity but to be required, in some cases, for the generation of cytotoxicity. Endogenous IL-4 production was observed and demonstrated to be important because neutralizing antiserum to IL-4 suppressed CTL development. In contrast to the effects of IL-4 when added later to the cultures, pulsing the lymphocytes with IL-4 before, or shortly after, exposure to antigen resulted in suppression of the CTL response. These results indicate that IL-4 has differentiative, proliferative, and suppressive effects on cell-mediated immune responses.     

Human Serum Promotes Osteogenic Differentiation of Human Dental Pulp Stem Cells In Vitro and In Vivo

Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS). FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS) is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.