Stem and stromal cell reconstitution of lethally irradiated mice following transplantation of hematopoietic tissue from donors of various ages

If the limited life span of hematopoietic tissues in vitro is due to a finite proliferative capacity of individual stem cells, one might expect tissues of young donors to possess a greater proliferative capacity and to contain a larger population of primitive stem cells than those of older donors. To test this hypothesis, we used 12- and 8-day spleen colony formation (CFU-s) to assay more and less primitive stem cell subpopulations of three murine hematopoietic tissues: fetal liver (FL) and weanling (WBM) and adult (ABM) bone marrow. Subsequently, the same assays and a stromal cell assay were performed on the bone marrow from groups of lethally irradiated mice reconstituted with these tissues. Comparison of the CFU-s content of the donor tissues revealed that FL contained a significantly greater proportion of primitive stem cells as evidenced by a (Day 12):(Day 8) CFU-s ratio of 3.0 +/- 1.0 as compared to 0.9 +/- 0.1 for WBM and ABM. In addition, at 21 weeks post-transplantation the CFU-s/femur values of the FL reconstituted group were significantly greater than those of the ABM and WBM reconstituted groups. These results suggest that fetal hematopoietic tissue contains a greater proportion of primitive stem cells and has a greater proliferative potential than hematopoietic tissue from older donors. No differences were seen in stromal cell reconstitution of the three experimental groups. In all cases, assayable fibroblast colony forming cells (CFU-f) remained at 20-40% of control values, even at 21 weeks postreconstitution.     

Comparative evaluation of mesenchymal stromal cells from umbilical cord and amniotic membrane in xeno-free conditions

Background

Within the past years, umbilical cord (UC) and amniotic membrane (AM) expanded in human platelet lysate (PL) have been found to become increasingly candidate of mesenchymal stromal cells (MSCs) in preclinical and clinical studies. Different sources of MSCs have different properties, and lead to different therapeutic applications. However, the similarity and differences between the AMMSCs and UCMSCs in PL remain unclear.

Results

In this study, we conduct a direct head-to-head comparison with regard to biological characteristics (morphology, immunophenotype, self-renewal capacity, and trilineage differentiation potential) and immunosuppression effects of AMMSCs and UCMSCs expanded in PL. Our results indicated that AMMSCs showed similar morphology, immunophenotype, proliferative capacity and colony efficiency with UCMSCs. Moreover, no significantly differences in osteogenic, chondrogenic and adipogenic differentiation potential were observed between the two types of cells. However, AMMSCs exhibited higher PGE₂ expression and IDO activity compared with UCMSCs when primed by IFN-γ and (or) TNF-α induction, and AMMSCs showed a higher inhibitory effect on PBMCs proliferation than UCMSCs.

Conclusion

The results suggest that AMMSCs expanded in PL showed similar morphology, immunophenotype, self-renewal capacity, and trilineage differentiation potential with UCMSCs. However, AMMSCs possessed superior immunosuppression effects in comparison with UCMSCs. These results suggest that AMMSCs in PL might be more suitable than UCMSCs for treatment of immune diseases. This work provides a novel insight into choosing the appropriate source of MSCs for treatment of immune diseases.

     

Epidermal stem cells: targets for carcinogenic chemicals

Epidermal stem cells long have been considered a target for carcinogenic chemicals, but these stem cells have never been identified or isolated. Toward this goal, this report examines two-stage carcinogenesis in light of the stem cell model for cellular replacement in the epidermis and considers characteristics that may be useful in the identification and the isolation of epidermal stem cells. Firstly, the carcinogenesis experiments in mice have indicated that the population of target cells normally remains in the epidermis for the life of the animal despite the continual cellular turnover. Hence, the slowly cycling (label-retaining) keratinocytes from the epidermis and hair follicles are potential targets. Secondly, the results of carcinogenesis experiments have also indicated that the target cells are necessarily ones with a high potential for proliferation relative the most of the proliferative population. The keratinocyte colony forming units (kCFU) from the epidermis of normal and treated adult mice are consequently a quantifiable indicator of proliferative potential and another possible target. Further application of the stem cell concepts of quiescence and of self-renewal is expected to yield additional tools for the identification and isolation of the epidermal targets for chemical carcinogens.     

Telomere shortening and chromosomal instability abrogates proliferation of adult but not embryonic neural stem cells

Chromosome integrity is essential for cell viability and, therefore, highly proliferative cell types require active telomere elongation mechanisms to grow indefinitely. Consistently, deletion of telomerase activity in a genetically modified mouse strain results in growth impairments in all highly proliferative cell populations analyzed so far. We show that telomere attrition dramatically impairs the in vitro proliferation of adult neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of telomerase-deficient adult mice. Reduced proliferation of postnatal neurogenic progenitors was also observed in vivo, in the absence of exogenous mitogenic stimulation. Strikingly, severe telomere erosion resulting in chromosomal abnormalities and nuclear accumulation of p53 did not affect the in vitro proliferative potential of embryonic NSCs. These results suggest that intrinsic differences exist between embryonic and adult neural progenitor cells in their response to telomere shortening, and that some populations of tissue-specific stem cells can bypass DNA damage check points.     

Macrophage colony stimulating factor (M-CSF) within cord blood sera may be partially responsible for the reduced proliferation of cord blood T cells

We show that there are differences in the soluble factors in cord blood (CB) and adult serum and that these differences play a role in T cell function. Thus, the mitogen and alloantigen-specific proliferative response of adult T cells was enhanced with increasing concentrations of adult serum and CB serum, but to a lesser extent with CB serum. In addition, proliferation of T cells induced by stimulation through the T cell receptor alone (via CD3 stimulation), could be enhanced with adult but not CB serum. However, CB serum enhanced the IL-2-specific proliferative response of pure T cells whereas adult serum did not. To determine whether there was an anti-inflammatory cytokine within CB serum which could induce these results, we assayed our serum samples for anti-inflammatory cytokines. IL-13 could not be detected in any serum sample, whereas IL-10 could be detected in adult but not CB serum (P < 0.002). However, there was a significant difference in the levels of macrophage colony stimulating factor (M-CSF) detected in adult and CB serum samples (P < 0.01). M-CSF was detected in 6/7 CB serum samples (mean +/- SD was 3.8 +/- 2.3 ng/ml) and 0/5 adult serum samples. Furthermore, anti-M-CSF antibody restored the reduced allo-response of T cells incubated in CB serum. Thus, M-CSF may act as a suppressor factor in CB serum. Whether this is sufficient to explain the lack of an allo-response by the foetus to the mother, or the reduced graft-versus-host disease when CB is used instead of bone marrow in stem cell transplantation, is yet to be determined.     

Effect of cryopreservation on biological and immunological properties of stem cells from apical papilla

Stem cells from apical papilla (SCAP) are a novel population of multipotent stem cells that, although similar to dental pulp stem cells, are a discrete source of dental stem cells. SCAP have potential roles in root development, apexogenesis, pulp/dentin regeneration, and bioroot engineering. However, procedures to store and preserve SCAP for future clinical applications have not been explored. In this study, we compared human freshly isolated SCAP (fSCAP) with cryopreserved SCAP (cSCAP) in terms of cell viability, colony‐forming efficiency, cell proliferation rate, multilineage differentiation potential, profiles of mesenchymal stem cell (MSC) markers, karyotype analysis, and immunological assays. cSCAP showed a similar viable cell ratio, colony‐forming efficiency, cell proliferation rate, multilineage differentiation potential, MSC surface markers, apoptotic rate, and G‐banded karyotype when compared to fSCAP. There was no significant difference between fSCAP and cSCAP with regard to immune properties. In addition, cSCAP of miniature pig possessed the similar proliferation rate, differentiation potential, and immunomodulatory function as seen in fSCAP. This study demonstrates that cryopreservation does not affect the biological and immunological properties of SCAP, supporting the feasibility of SCAP cryopreservation in nitrogen. J. Cell. Physiol. 223: 415–422, 2010. © 2010 Wiley‐Liss, Inc.     

Correlation between proliferative activity and cellular thickness of human mesenchymal stem cells

A cell’s shape is known to be related to its proliferative activity. In particular, large and flat mammalian adult stem cells seem to show slow proliferation, however using quantitative analysis to prove the phenomenon is difficult. We measured the proliferation and cellular thickness of human mesenchymal stem cells (MSCs) by atomic force microscopy and found that MSCs with high proliferative activity were thick while those with low proliferative activity were thin, even though these MSCs were early passage cells. Further, low proliferative MSCs contained many senescence-associated β-galactosidase positive cells together with high senescence-associated gene expression. These findings suggest that the measurement of cellular thickness is useful for estimating the proliferative activity of human MSCs and is expected to be a practical tool for MSC applications in regenerative medicine.     

HEMOPOIETIC STEM CELL PROLIFERATION AND MIGRATION FOLLOWING BORDETELLA PERTUSSIS VACCINE

The ability of a single injection of killed, intact bacteria to effect an increase in the proliferative rate of hemopoietic stem cells was studied. The total numbers of colony forming units in bone marrow, spleen and peripheral blood as well as the proportion of CFU in cycle was assessed. Splenic CFU were observed to rise exponentially due initially to in situ proliferation and later to proliferation in bone marrow with migration via the blood to the spleen. The results are discussed in the light of current concepts of stem cell regulation.     

A DEFICIENCY OF HEMATOPOIETIC STEM CELLS IN STEEL MICE

In the present study, population sizes of high self-renewal potential stem cells, i.e. colony forming units (CFU), and low self-renewal potential stem cells, i.e. transient endogenous colony forming units (TE-CFU) in Sl/Sl^d mice and their normal congenic littermates were measured and compared. By correcting for differences in the seeding efficiency ‘f’, it was possible to demonstrate that Sl/Sl^d mice suffer a deficiency of both stem cell populations. Therefore, it is concluded that the defective stromal tissue of the Sl/Sl^d mouse does not support normal size stem cell populations. However, as noted in the discussion, it remains an open question as to whether the defective stromal tissue supports normal erythroid differentiation at the stem cell level.     

Elimination of red blood cells from marrow suspension prior to transplantation using polysaccharide-ditrizoate gradient centrifugation. Experimental studies

In a search of alternative techniques of red cell depletion from the marrow prior AB0 incompatible transplantation we got interested in B?yum technique of polysaccharide-ditrizoate gradient centrifugation for isolation of mononuclear cells. This method might be used for the purpose of red cell depletion only if it preserves haemopoiesis reconstituting stem cells. Whether this is the case was tested using murine model of lethally irradiated donors. Polysaccharide-ditrizoate gradient isolated mononuclear cells contained 100% spleen colony forming stem cells and possessed the same bone marrow and spleen reconstitutive potential as non-manipulated marrow cells. Therefore, this method may be considered for potential clinical application.     

Comparison of umbilical cord tissue-derived mesenchymal stromal cells isolated from cryopreserved material and extracted by explantation and digestion methods utilizing a split manufacturing model

Background aimsUmbilical cord (UC) tissue is recognized as an advantageous source of mesenchymal stromal cells (MSCs), whose therapeutic properties are being actively evaluated in pre-clinical and clinical trials. In recognition of its potential value, storage of UC tissue or cells from UC tissue in newborn stem cell banks is now commonplace; however, strategies for isolating UC-derived MSCs (UCMSCs) from UC tissue have not been standardized. The majority of newborn stem cell banks take one of two approaches to cord tissue processing and cryopreservation: enzymatic digestion of the fresh tissue with cryopreservation of the subsequent cell suspension or cryopreservation of the tissue as a composite whole with later, post-thaw isolation of cells by explantation. Evaluation of UCMSCs derived by these two principal preparation and cryopreservation strategies is important to understanding whether the methods currently employed by newborn stem cell banks retain the desirable clinical attributes of UC cells.MethodsUCMSCs were isolated from 10 UC tissue samples by both explantation and enzymatic digestion methods to allow for comparison of cells from the same donor. Cell isolates from both methods were compared pre- and post-cryopreservation as well as after serial passaging. Cell viability, morphology, growth kinetics, immunophenotype, cytokine secretion and differentiation capacity were evaluated.ResultsUCMSCs could be derived from fresh UC tissue by both explantation and digestion methods and from thawed UC tissue by explantation. Initial cell populations isolated by digestion were heterogeneous and took longer to enrich for UCMSCs in culture than populations obtained by explantation. However, once isolated and enriched, UCMSCs obtained by either method showed no significant difference in viability, morphology, rate of proliferation, surface marker expression, levels of cytokine secretion or differentiation capacity.ConclusionsDerivation of UCMSCs by explantation after thawing UC cryopreserved as a composite tissue may be favorable in terms of initial purity and number of cells achievable by a specific passage. However, we observed no evidence of functional difference between UCMSCs derived by explanation or digestion, suggesting that cells isolated from cryopreserved material obtained by either method maintain their therapeutic properties.     

人成体心源间充质样细胞表型分析及向心脏谱系诱导分化潜能的研究

目的探讨成人心源性间充质样细胞 (CDMCs)的分子表型及向心脏谱系的分化潜能。方法实验分为:不同培养时间CDMCs (第3、5、7代),并以脐带间充质干细胞 (UCMSCs)为对照。分析各细胞分子表型并向心脏谱系诱导分化。显微镜观察细胞形态;计算生长倍增时间并绘制细胞生长曲线;流式细胞术分析表面标志抗原表达;实时定量PCR和Western blot分别测干细胞多能分子及组织特异性分子mRNA和蛋白表达。结果采用重复测量资料方差分析、单因素方差分析和配对t检验。结果 CDMCs具有UCMSCs形态特征与增殖能力,体外培养1 ~ 7 d,与UCMSCs比较,P3、5、7代CDMCs增殖能力差异无统计学意义 (P> 0.05)。与UCMSCs相比,不同培养时间CDMCs表面标志抗原 (CD90)表达 (冻存前:97.13%±2.00%比59.87%±34.14%、38.83%±11.04%、34.77±14.78%;冻存后:99.83%±0.17%比56.00%±19.47%、47.48±11.88%、41.15±8.68%)降低(P< 0.05)。与UCMSCs相比,不同培养时间CDMCs中Rex1 (0.00±0.00比0.68±0.50、0.29±0.17、0.38±0.50)、Oct3/4 (1.00±0.02比5.28±0.78、3.88±0.95、3.63±0.34)、Nanog(1.00±0.16比7.57±4.69、5.40±3.58、5.34±0.76)以及心脏特异转录因子Nkx2.5 (1.00±0.12比30.60±22.43、19.69±9.65、8.82±4.94)、Gata4 (1.00±0.85比60467±25266、44350±25800、35067±23113)表达均增高,差异有统计学意义 (P均< 0.05)。与诱导前比较,向心肌诱导分化15 d后,不同培养时间CDMCs中cTnT蛋白表达水平 (0.40±0.13比0.98±0.16、0.38±0.18 比0.69±0.15、0.17±0.11比0.70±0.17)增高 (P< 0.05)。结论 CDMCs不仅具备部分干细胞和间充质细胞表型,还具有心脏组织特异性。其具备心脏谱系分化潜能,心肌细胞分化能力可能优于UCMSCs。     

AcSDKP抑制体外培养条件下人骨髓间充质干细胞的增殖

N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(N-acetyl-seryl-aspartyl-lysyl-proline,AcSDKP)是一种具有生理调控活性的四肽因子,对造血干/祖细胞增殖具有抑制作用。本研究采用集落形成实验、甲基偶氮唑盐(MTT)比色法、细胞分裂指数测定等方法,考察了AcSDKP对体外培养的人骨髓间充质干细胞(mesenchymal stem cell,MSC)增殖的影响。结果显示,在AcSDKP浓度为1×10-12mol／L-1×10-9mol／L的培养体系中,人骨髓MSC集落生成率和大小、活力细胞数和分裂指数均降低,最大效应浓度为1×10-11mol／L。以上实验结果表明,在体外培养条件下,一定浓度的AcSDKP对人骨髓MSC 的增殖具有抑制作用。     

Different recovery patterns of mouse haemopoietic stem cells in response to cytotoxic agents

The response and subsequent recovery of mouse haemopoietic progenitor cells (spleen colony forming cells and agar colony forming cells) has been studied following two cytotoxic agents. Busulphan was administered to normal mice and vinblastine to mice where the progenitor cell proliferation rate had been increased by a period of continuous γ-irradiation. With both these agents there is a difference between the response of the spleen colony forming cells and the agar colony forming cells during the first five days. They then recover together, but much more slowly after busulphan than after vinblastine even though their proliferation rate is increased. The rate of progenitor cell recovery after busulphan is increased if the progenitor cells are depleted further by vinblastine. However, methotrexate, which severely depletes the peripheral blood count and bone marrow cellularity but not the progenitor cells, has no effect on the recovery following busulphan. These results suggest that following cytotoxic agents the agar colony forming cells (“committed” stem cells) are not self-maintaining but are dependent on a supply of cells from the pluripotential spleen colony forming cells. In addition it appears that the depletion of the progenitor cells of the bone marrow and not the depletion of the maturing cells, provides a stimulus for stem cell recovery.     

Retinal stem/progenitor properties of iris pigment epithelial cells

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina and ciliary epithelium of the adult eye. Here, we first demonstrate the characterization of neural stem/progenitors in postnatal iris pigment epithelial (IPE) cells. Pure isolated IPE cells could form spheres that contained cells expressing retinal progenitor markers in non-adherent culture. The spheres grew by cell proliferation, as indicated by bromodeoxyuridine incorporation. When attached to laminin, the spheres forming IPE derived cells were able to exhibit neural phenotypes, including retinal-specific neurons. When co-cultured with embryonic retinal cells, or grafted into embryonic retina in vivo, the IPE cells could also display the phenotypes of photoreceptor neurons and Muller glia. Our results suggest that the IPE derived cells have retinal stem/progenitor properties and neurogenic potential without gene transfer, thereby providing a novel potential source for both basic stem cell biology and therapeutic applications for retinal diseases.     

间充质干细胞的细胞成像技术比较与应用

间充质干细胞是一类具有强大增殖、多向分化潜能和免疫调节能力的多功能细胞,研究显示间充质干细胞移植可能治疗多种难治性疾病,例如帕金森病、脊髓损伤以及肿瘤等。但是,人们对移植后的细胞在宿主内的存活、分布、增殖、分化、免疫排斥反应以及成瘤特性等问题尚不清楚,所以许多疾病经过细胞移植治疗后的进展及转归情况仍难以获得确切的科学证据。而细胞成像技术(包括放射性核素成像、超声成像、磁共振成像以及光学成像)可以在体外或者体内实现对间充质干细胞实时、无创的示踪,在以间充质干细胞为研究基础的细胞移植治疗和细胞组织再生的医学领域里有着巨大的应用潜力。该文综述近十年来细胞成像技术应用于示踪间充质干细胞移植疗法的研究进展,旨在比较当下多种热门细胞成像技术的优劣,进而找寻更合适的干细胞示踪策略,为干细胞移植治疗的基础和临床研究提供进一步的理论证据支持和研究思路。     

Reovirus safety study for proliferation and differentiation of human adipose-derived mesenchymal stem cells

Naturally occurring reoviruses are live replication-proficient viruses specifically infecting human cancer cells while sparing the normal counterparts. Stem cells can be highly susceptible to viral infection due to their innate high proliferation potential and other active signaling pathways of cells that might be involved in viral tropism. In the previous study, we showed that reoviruses could adversely affect murine embryonic stem cells’ integrity in vitro and in vivo. Oncolytic viruses, delivered systemically face many hurdles that also impede their localization and infection of, metastatic tumors, due to a variety of immune and physical barriers. To overcome such hurdles to systemic delivery, several studies supported the idea that certain types of cells, including mesenchymal stem cells, might play a role as cell carriers for oncolytic viruses. Thus, it would be interesting to examine whether human adult stem cells such as human adipose-derived mesenchymal stem cells could be saved by the reoviral challenge. In this study, we report that biological activities such as proliferation and multipotency of human adipose-derived stem cells are not affected by wild-type reovirus challenge as evidenced by survival, osteogenic and adipogenic differentiation potential assays following treatment with reoviruses. Therefore, unlike murine embryonic stem cells, our study strongly suggests that human adipose-derived adult stem cells could be spared in vivo during wild-type reoviral anti-cancer therapeutics in a clinical setting. Furthermore, the results support the possible clinical use of human adipose-derived stem cells as an effective cell carrier of oncolytic reovirus to maximize their tumor tropism and anti-tumor activity.