Osteogenic and adipogenic capacity of fibroblast-like progenitor cells derived frow human fetal liver

The study of differentiation potential of multiponent stromal progenitor cells (PCs) in embryogenesis is a crucial issue for understanding their biology and role in tissue regeneration of an adult organism. In this study in monolayer culture there were investigated osteogenic and adipogenic capacities of fibroblast-like PCs derived from human fetal liver of 8-11 gestation weeks before and after exposure to cryoprotectant dimethyl sulphoxide (DMSO). It was shown that the primary suspension of human fetal liver cells included immature stromal fibroblast-like PCs which were able to be induced into osteogenic and adipogenic differentiation. A short-time exposure of freshly isolated human fetal liver cells to cryoprotectant DMSO led to altering properties of the fibroblast-like PCs. Under subculture conditions, it was found an increase in the number of fibroblast-like PCs which were able to be induced to osteogenic differentiation in vitro. The established fact of DMSO influence on the differentiation capacity of fetal fibroblast-like PCs is necessary to take into consideration while developing cryopreservation methods for stem cells.     

Osteogenic and adipogenic capacity of fibroblast-like progenitor cells derived from human fetal liver

The study of the differentiation potential of multipotent stromal progenitor cells (PC) in embryogenesis is a crucial issue for understanding their biology and role in the tissue regeneration of an adult organism. In this study, in monolayer culture, osteogenic and adipogenic potencies of fibroblast-like PCs derived from human fetal liver of 8–11 gestation weeks were investigated before and after exposure to cryoprotectant dimethyl sulphoxide (DMSO). It was shown that the primary suspension of human fetal liver cells includes immature stromal fibroblast-like PCs, which were able to induce osteogenic and adipogenic differentiation. The short-term exposure of recently isolated human fetal liver cells to cryoprotectant DMSO led to alterations in the properties of fibroblast-like PCs. Under subculture conditions, an increase in the number of fibroblast-like PCs capable of inducing osteogenic differentiation in vitro was discovered. It is necessary to take this established fact of DMSO influence on the differentiation capacity of fetal fibroblast-like PCs into consideration when developing cryopreservation methods for stem cells.     

Boron increases the cell viability of mesenchymal stem cells after long-term cryopreservation

The field of stem-cell biology has emerged as a key technology for the treatment of various disorders and tissue regeneration applications. However, a major problem remains in clinical practice, which is the question of whether stem cells preserve their self-renewal and differentiation potential in the culture conditions or not. In the current study, effects of boron on the cryopreservation of human tooth germ stem cells (hTGSCs) were evaluated for the first time. The impacts of various boron concentrations (sodium pentaborate pentahydrate (NaB)) were tested on characterized hTGSCs viability for different time intervals (24, 48, and 72 h). 20 μg/ml NaB with lower Me₂SO concentration was found to display positive effects on hTGSCs during repeated freezing and defrosting cycles, and long-term cryopreservation. After thawing, cells were analyzed for their surface antigens and differentiation capacity. hTGSCs were successfully cryopreserved without any change in their mesenchymal stem cell characteristics as they were treated with boron containing freezing medium. In addition, fatty acid composition was examined to demonstrate membrane fatty acid profiles after freeze-thawing. Besides, NaB treatment extended osteogenic and chondrogenic differentiation of hTGSCs remarkably after long-term cryopreservation with respect to control groups. The study clearly suggests that NaB has a protective role on the survival of hTGSCs in short- and long-term cryopreservation. Due to the possible storage of hTGSCs at early ages, development of a functional and reliable cryopreservation media can be designed as a future solution to the dental stem cell banking.     

The osmotic characteristics of human fetal liver-derived hematopoietic stem cell candidates

Hematopoietic stem cells derived from fetal liver have promising therapeutic potential for allotransplantation but require a specific protocol to minimize the damage produced by cryopreservation procedures. In this study, a fundamental approach was applied for designing a cell preservation protocol. To this end, the biophysical characteristics that describe the osmotic reaction of CD34(+)CD38(-) human fetal liver stem cell candidates were studied using fluorescent microscopy. The osmotically inactive volume of the stem cell candidates was determined as 48% of the isotonic volume. The permeability coefficients for water and Me(2)SO were determined at T = +22 degree C: L(p) = 0.27 +/- 0.03 microm x min(-1)atm(-1), P(Me(2)SO)) = 2.09 +/- 0.85 x 10 (-4) cm x min(-1), sigma (Me(2)SO)) = 0.63 +/- 0.03 and at T = +12 degree C: L(p) = 0.15 +/-0.02 microm x min(-1)atm(-1), P(Me(2)SO)) = 6.44 +/-1.42 x 10 (-5) cm x min(-1), sigma (Me(2)SO)) = 0.46 +/- 0.05. The results obtained suggest that post-hypertonic and hypotonic stress are the possible reasons for damage to a CD34(+)CD38(-) cell during the cryopreservation procedure.     

Evaluation of intracellular and extracellular trehalose as a cryoprotectant of stem cells obtained from umbilical cord blood

Cord blood is a source of hematopoietic stem cells used in transplantation in which hematopoietic reconstitution is necessary. This transplant modality requires the cryopreservation of hematopoietic stem cells (HSCs). Dimethyl sulfoxide has been used as a cryoprotectant (CPA) in the cryopreservation of HSCs; however, it has been demonstrated that Me₂SO exhibits toxic side effects to the human body. Due to its stability upon freezing, disaccharides such as trehalose have been investigated as a cryoprotectant. This study investigated the hypothesis that a cryopreservation solution containing intracellular and extracellular trehalose improves the recovery of stem cells after cryopreservation. After thawing, the cells were tested for their viability using the 7AAD stain, CD45+/CD34+ cells were assessed using flow cytometry and the MTT viability assay, and the proportion of hematopoietic progenitor cells was measured using the CFU assay. Our results showed the effectiveness of the solution containing intracellular and extracellular trehalose in the cryopreservation of cord blood cells, demonstrating that trehalose may be an optimal cryoprotectant when present both inside and outside of cells.     

Nonhematopoietic stem cells of fetal origin--how much of today's enthusiasm will pass the time test?

Stem cells originating at fetal age are for many reasons superior as a material for the regenerative medicine purposes, when compared to their adult counterparts. While hematopoietic cells, isolated from fetal liver or cord blood, have been well known for a long time and have passed practical tests as clinical transplantation material, the non-hematopoietic cells are newly recognized, and the knowledge of their phenotype and differentiation potential is rather insufficient. We, and the others, have identified a subpopulation of cord blood cells phenotypically different from hematopoietic cells (CD34-, CD45-, CD29+, CD44+, CD51+, CD105+, SH-2, SH-3), in vitro plastic adherent, and capable of multilineage differentiation. The other candidates for multipotential stem cells are cells extracted from umbilical cord or placental tissue. The preliminary observations suggest, that these cells, phenotypically similar to the nonhematopoietic cord blood cells, are capable of extensive replication in vitro and of multilineage differentiation into a variety of tissues including cardiac muscle, bone and cartilage, adipocytes, and nerve cells. The other possible medical applications include "rejuvenation" of selected tissues and systems in senile patients, and therapeutical cloning - for both purposes, cells at the fetal stage of genetic regulation may be more useful than cells collected from adult donors. There is still, however, a high level of uncertainty concerning future medical applications of fetal stem cells. Their numbers and characteristics may differ from the preliminary observations, and their behavior in vivo may not fulfill the expectations originating from the in vitro studies. Finally, the autologous applications of stem cells collected at the stage of birth may need the involvement of technical and financial resources for the storage of frozen cell samples throughout the period of life of their potential user. Such procedure seems possible from technical point of view, but may be inadequately substantiated by the eventual advantages.     

Cells isolated from cryopreserved dental follicle display similar characteristics to cryopreserved dental follicle cells

Dental follicle tissue is a promising resource of mesenchymal stem cells for cytotherapeutic approaches and tissue engineering applications. There are two procedures for banking of human dental follicle stem cells have been reported. Conventional method requires cell isolation, expansion and immediate cryopreservation. Whereas dental follicle stem cells can be isolated from cryopreserved dental follicle fragments. The aim of this study was to compare the characteristics of dental follicle cells isolated from cryopreserved fragments (DFCs-CF) with dental follicle cells recovered from cryopreserved cells (DFCs-CC). Dental follicle fragments obtained after mechanical disaggregation were divided into two parts, with one part maintained in culture, while another part underwent cryopreservation. Dental follicle fragments and dental follicle cells from fresh tissue were stored in liquid nitrogen for 3 months. After thawing, the isolation, morphology, proliferation, cell cycle, colony-forming-unit ability, stemness-related marker expression, apoptosis, and multi-lineage differentiation potential of DFCs-CF were tested compared with DFCs-CC. DFCs-CF expressed mesenchymal stem cells marker, proliferated well, showed similar levels of mRNA for stemness- and apoptosis-related genes and exhibited the capacity of multi-lineage differentiation similar to those of DFCs-CC. These results imply that cryopreservation of dental follicle fragments is an effective banking method for isolation of dental follicle cells.     

Human mesenchymal stem cells - current trends and future prospective

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton''s jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.     

Hepatic differentiation from human mesenchymal stem cells on a novel nanofiber scaffold

The emerging fields of tissue engineering and biomaterials have begun to provide potential treatment options for liver failure. The goal of the present study is to investigate the ability of a poly L-lactic acid (PLLA) nanofiber scaffold to support and enhance hepatic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). A scaffold composed of poly L-lactic acid and collagen was fabricated by the electrospinning technique. After characterizing isolated hMSCs, they were seeded onto PLLA nanofiber scaffolds and induced to differentiate into a hepatocyte lineage. The mRNA levels and protein expression of several important hepatic genes were determined using RT-PCR, immunocytochemistry and ELISA. Flow cytometry revealed that the isolated bone marrow-derived stem cells were positive for hMSC-specific markers CD73, CD44, CD105 and CD166 and negative for hematopoietic markers CD34 and CD45. The differentiation of these stem cells into adipocytes and osteoblasts demonstrated their multipotency. Scanning electron microscopy showed adherence of cells in the nanofiber scaffold during differentiation towards hepatocytes. Our results showed that expression levels of liver-specific markers such as albumin, α-fetoprotein, and cytokeratins 8 and 18 were higher in differentiated cells on the nanofibers than when cultured on plates. Importantly, liver functioning serum proteins, albumin and α-1 antitrypsin were secreted into the culture medium at higher levels by the differentiated cells on the nanofibers than on the plates, demonstrating that our nanofibrous scaffolds promoted and enhanced hepatic differentiation under our culture conditions. Our results show that the engineered PLLA nanofibrous scaffold is a conducive matrix for the differentiation of MSCs into functional hepatocyte-like cells. This represents the first step for the use of this nanofibrous scaffold for culture and differentiation of stem cells that may be employed for tissue engineering and cell-based therapy applications.     

一种无血清冻存液在猪胎儿成纤维细胞上的应用研究

在猪胎儿成纤维细胞(porcine fetal fibroblasts, PFF)冻存过程中,血清品质常常制约着细胞的冻存效果。为了解决这个问题,本研究旨在开发一种无血清冻存液应用于猪胎儿成纤维细胞冻存。用3种不同冻存液冻存猪胎儿成纤维细胞,每种冻存10管。冻存30 d后复苏细胞,测定冻存细胞存活率,细胞增殖活力以及电转后细胞活性。结果显示:自制无血清细胞冻存液,冻存猪胎儿成纤维细胞后存活率达95.33%;细胞增殖活力以及电转后细胞活性均显著高于标准胎牛血清冻存液(p<0.05),与特级胎牛血清冻存液效果相当(p>0.05)。因此,自制冻存液冻存猪胎儿成纤维细胞效果稳定,能够替代含血清冻存液,有良好的推广应用前景。     

Multipotent mesenchymal stem cells from amniotic fluid originate neural precursors with functional voltage-gated sodium channels

Background aimsAmniotic fluid (AF) contains stem cells with high proliferative and differentiative potential that might be an attractive source of multipotent stem cells. We investigated whether human AF contains mesenchymal stem cells (MSC) and evaluated their phenotypic characteristics and differentiation potential in vitro.MethodsAF was harvested during routine pre-natal amniocentesis at 14–16 weeks of pregnancy. AF sample pellets were plated in α-minimum essential medium (MEM) with 10% fetal bovine serum (FBS). We evaluated cellular growth, immunophenotype, stemness markers and differentiative potential during in vitro expansion. Neural progenitor maintenance medium (NPMM), a medium normally used for the growth and maintenance of neural stem cells, containing hFGF, hEGF and NSF-1, was used for neural induction.ResultsTwenty-seven AF samples were collected and primary cells, obtained from samples containing more than 6 mL AF, had MSC characteristics. AF MSC showed high proliferative potential, were positive for CD90, CD105, CD29, CD44, CD73 and CD166, showed Oct-4 and Nanog molecular and protein expression, and differentiated into osteoblasts, adypocytes and chondrocytes. The NPMM-cultured cells expressed neural markers and increased Na⁺ channel density and channel inactivation rate, making the tetrodotoxin (TTX)-sensitive channels more kinetically similar to native neuronal voltage-gated Na⁺ channels.ConclusionsThese data suggest that AF is an important multipotent stem cell source with a high proliferative potential able to originate potential precursors of functional neurons.     

Structure and function of human fetal endocrine pancreas before and after cryopreservation

Human fetal pancreatic glands obtained from 31 consecutive prostaglandin-induced abortions were examined with respect to light microscopic structure and insulin content and release before and after cryopreservation. The crown-heel lengths of the fetuses ranged from 12 to 34 cm. Minced pancreatic fragments about 2 mm³ in size were cultured overnight in RPMI 1640 medium plus 10% fetal calf serum. The explants were incubated at 0 °C for 20 min in Hanks' solution containing 1 M Me₂SO and subsequently cooled at 0.3 °C/min to ?70 °C before rapid quenching in liquid nitrogen. After storage for 4–150 days at ?196 °C the pancreatic fragments were rapidly thawed and suspended in RPMI 1640 (10% calf serum) for another overnight culture.After cryopreservation there was some morphological deterioration of the fetal pancreas. Before cryopreservation 13 of the fetal glands responded with a significant insulin release to an acute glucose plus theophylline challenge, while after cryopreservation 16 glands responded.Although cryopreservation lowered the insulin response there was a strong statistical correlation between the response obtained before and after freezing (P < 0.001). No correlation could be demonstrated between the insulin response and crown-heel length either before or after freezing. There was no obvious effect of cryopreservation on the pancreatic insulin content which showed a significant correlation with the crown—heel length both before and after freezing.It is concluded that cryopreservation of human fetal endocrine pancreas preserves the viability of the B cells. These observations provide a basis for further exploration of the suitability of human fetal pancreas for clinical transplantation.     

Cultured buffalo umbilical cord matrix cells exhibit characteristics of multipotent mesenchymal stem cells

Recent findings have demonstrated umbilical cord, previously considered as a biomedical waste, as a source of stem cells with promising therapeutic applications in human as well as livestock species. The present study was carried out to isolate the umbilical cord matrix cells and culture for a prolonged period, cryopreserve these cells and test their post-thaw viability, characterize these cells for expression of stem cell markers and differentiation potential in vitro. The intact umbilical cord was taken out of the amniotic sac of a fetus and then incised longitudinally to remove umbilical vessels. Wharton’s jelly containing tissue was diced into small pieces and placed in tiny drops of re-calcified buffalo plasma for establishing their primary culture. Confluent primary culture was trypsinized and passaged with a split ratio of 1:2 for multiplication of cells. Cryopreservation of cells was performed at three different passages in cryopreservation medium containing 15%, 20% and 25% fetal bovine serum (FBS). A significant increase in post-thaw viability was observed in cells cryopreserved in freezing medium with higher concentration of FBS. After re-culturing, frozen-thawed cells started adhering, and spike formation occurred within 4–6 h with similar morphology to their parent representative cultures. The normal karyotype and positive expression of alkaline phosphatase and pluripotency genes OCT4, NANOG and SOX2 were observed at different passages of culture. When induced, these cells differentiated into adipogenic and osteogenic cells as confirmed by oil red O and alizarin red stains, respectively. This study indicates that buffalo umbilical cord matrix cells have stemness properties with mesenchymal lineage restricted differentiation and limited proliferation potential in vitro.     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

Serum-free cryopreservation of human amniotic epithelial cells before and after isolation from their natural scaffold

Amniotic epithelial cells are a promising source for stem cell-based therapy through their potential capacity to differentiate into the cell lineages of all three germ layers. Long-term preservation is necessary to have a ready-to-use source of stem cells, when required. Reduced differentiation capability, decrease of viability and use of fetal bovine serum (FBS) are three drawbacks of clinical application of cryopreserved stem cells. In this study, we used human amniotic fluid instead of animal serum, and evaluated viability and multipotency of amniotic epithelial cells after cryopreservation in suspension and compared with those cryopreserved on their natural scaffold (in situ cryopreservation). There was no significant difference in viability of the cells cryopreserved in amniotic fluid and FBS. Also, the same results were achieved for expression of pluripotency marker OCT-4 when FBS was replaced by amniotic fluid in the samples with the same cryoprotectant. The cells cryopreserved in presence of scaffold had a higher level of viability compared to the cells cryopreserved in suspension. Although, the number of the cells expressed OCT-4 significantly decreased within cryopreservation in suspension, no decrease in expression of OCT-4 was observed when the cells cryopreserved with their natural scaffold. Upon culturing of post-thawed cells in specific lineage differentiating mediums, the markers of neuronal, hepatic, cardiomyocytic and pancreatic were found in differentiated cells. These results show that replacement of FBS by amniotic fluid and in situ cryopreservation of amniotic epithelial cells is an effective approach to overcome limitations related to long-term preservation including differentiation during cryopreservation and decrease of viability.     

Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo

In this study, we isolated CD31(-), CD34(-), CD106(-) (VCAM-1(-)), and fetal liver kinase(+) (Flk1(+)) cells from adipose tissue. These cells can be induced to differentiate into cells of osteogenic and adipogenic lineages in vitro and were termed adipose derived adult stem cells (ADAS cells). We also showed that they have characteristics of endothelial progenitor cells. In vitro, ADAS cells expressed endothelial markers when cultured with VEGF. In vivo, ADAS cells can differentiate in response to local cues into endothelial cells that contributed to neoangiogenesis in hindlimb ischemia models. PI3 kinase inhibitor LY294002 blocked the differentiation of ADAS cells into endothelial cells in vitro. Because ADAS cells can be expanded in culture without obvious senescence for more than 20 population doublings, they may be a potential source of endothelial cells for cellular pro-angiogenic therapies.     

Primary colonospheres maintain stem cell-like key features after cryopreservation

The development of efficient and repeatable protocols for biobanking and prolonged storage of cancer stem cells (CSCs), with minimum alterations in biological function, is valuable and desired, particularly for retrospective analysis and clinical applications. In particular, data regarding the effect of cryopreservation on CSCs's functional features is scarce. In this regard, few studies have been shown that 3D spheroid structures, which enriched for CSCs, can keep their biological phenotype and genetic profiles. Here, for the first time, we present data on cryopreservation of CT-26 colonospheres, with the focus on essential stem cell-like properties after thawing. Tumor biopsy-derived colonospheres were frozen in standard freezing media (90% fetal bovine serum + 10% dimethyl sulfoxide) and stored in liquid nitrogen for 10 months. Then, cryopreservation effect on preservation of CSCs-related features was verified using real-time polymerase chain reaction for evaluation of stemness genes and flow cytometry for the putative colorectal CSC surface biomarkers. The self-renewal capacity of thawed spheres was also compared with their fresh counterparts using serial formation assay. Finally, tumorigenic capacity of both groups was evaluated in immunocompetence mouse model. Our data indicated that postthawed colonospheres had high viability without drastic alteration in biological and structural features and maintained self-renewal potential after sequential passages. Real-time analysis showed that both fresh and frozen colonospheres displayed similar expression pattern for key stemness genes: SOX2 and OCT4. Cryopreserved spheroids expressed CD133, CD166, and DCLK1 CSCs surface biomarkers at elevated levels when compared with parental as non-cryopreserved counterparts. Our electron scanning microscopy micrographs clearly demonstrated that postthawed colonospheres retain their integrity and cell surface morphology and characteristics. We also found that both fresh and frozen spheroids were equally tumorigenic. This study represented an effective strategy for reliable storage of intact CT-26 colonospheres; this can provide researchers with a functionally reliable repository of murine colorectal CSCs for their future CSCs projects.     

人滑膜间充质干细胞与人脐带间充质干细胞的生物学性状比较

该文旨在比较人滑膜间充质干细胞(human synovial mesenchymal stem cells,hSMSCs)与人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)的生物学性状.流式细胞仪鉴定hSMSCs和hUC-MSCs.比较两种间...