Multipotent mesenchymal stromal cells (MSCs) are primitive cells capable of restoring damaged mesenchyme and with the ability to differentiate into mature cells of bone, cartilage, muscle, fat, nerve or fibrous tissues. MSCs are therefore good candidates for applications in regenerative medicine and cell based therapy. They regenerate through self-renewal, differentiational capacity, immune modulation and secretion of bioactive molecules. Authors present a review of MSCs applications in otorhinolaryngology. The major interest is focused on phonosurgery, sensorineural deafness and reconstruction of large tissue defects with bone, cartilage or soft tissue replacement. Current evidence of MSCs treatment efficacy in otorhinolaryngology is based on animal models. The true impact on clinical treatment will not be known until clinical studies prove functional outcomes in human medicine.

• MeSH
• biologické modely * MeSH
• buněčná diferenciace fyziologie   MeSH
• chrupavka cytologie   fyziologie   MeSH
• kosti a kostní tkáň cytologie   fyziologie   MeSH
• lidé MeSH
• mezenchymální kmenové buňky fyziologie   MeSH
• nervová tkáň cytologie   fyziologie   MeSH
• otorinolaryngologie metody   MeSH
• regenerativní lékařství metody   MeSH
• řízená tkáňová regenerace metody   MeSH
• transplantace mezenchymálních kmenových buněk metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

• MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• chronické selhání ledvin MeSH
• glomerulus růst a vývoj   MeSH
• modely u zvířat MeSH
• multipotentní kmenové buňky klasifikace   MeSH
• řízená tkáňová regenerace MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

Canal wall down mastoidectomy is one of the most effective treatments for cholesteatoma. However, it results in anatomical changes in the external and middle ear with a negative impact on the patient's quality of life. To provide complete closure of the mastoid cavity and normalize the anatomy of the middle and external ear, we used human multipotent mesenchymal stromal cells (hMSCs), GMP grade, in a guinea pig model. A method for preparing a biomaterial composed of hMSCs, hydroxyapatite, and tissue glue was developed. Animals from the treated group were implanted with biomaterial composed of hydroxyapatite and hMSCs, while animals in the control group received hydroxyapatite alone. When compared to controls, the group implanted with hMSCs showed a significantly higher ratio of new bone formation (p = 0.00174), as well as a significantly higher volume percentage of new immature bone (p = 0.00166). Our results proved a beneficial effect of hMSCs on temporal bone formation and provided a promising tool to improve the quality of life of patients after canal wall down mastoidectomy by hMSC implantation.

• MeSH
• biologické markery metabolismus   MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   MeSH
• modely nemocí na zvířatech MeSH
• morčata MeSH
• multipotentní kmenové buňky cytologie   MeSH
• počítačová rentgenová tomografie MeSH
• pooperační péče * MeSH
• spánková kost diagnostické zobrazování   patologie   chirurgie   MeSH
• transplantace mezenchymálních kmenových buněk * MeSH
• vnitřní ucho patologie   MeSH
• zánět patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• morčata MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Multipotent mesenchymal stromal cells (MSCs) can be considered an accessible therapeutic tool for regenerative medicine. Here, we compared the growth kinetics, immunophenotypic and immunomodulatory properties, gene expression and secretome profile of MSCs derived from human adult bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and Wharton's jelly (WJ-MSCs) cultured in clinically-relevant conditions, with the focus on the neuroregenerative potential. All the cell types were positive for CD10/CD29/CD44/CD73/CD90/CD105/HLA-ABC and negative for CD14/CD45/CD235a/CD271/HLA-DR/VEGFR2 markers, but they differed in the expression of CD34/CD133/CD146/SSEA-4/MSCA-1/CD271/HLA-DR markers. BM-MSCs displayed the highest immunomodulatory activity compared to AT- and WJ-MSCs. On the other hand, BM-MSCs secreted the lower content and had the lower gene expression of neurotrophic growth factors compared to other cell lines, which may be caused by the higher sensitivity of BM-MSCs to nutrient limitations. Despite the differences in growth factor secretion, the MSC secretome derived from all cell sources had a pronounced neurotrophic potential to stimulate the neurite outgrowth of DRG-neurons and reduce the cell death of neural stem/progenitor cells after H2O2 treatment. Overall, our study provides important information for the transfer of basic MSC research towards clinical-grade manufacturing and therapeutic applications.

• MeSH
• buněčná diferenciace * MeSH
• buňky kostní dřeně cytologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   metabolismus   MeSH
• nervové kmenové buňky cytologie   metabolismus   MeSH
• proliferace buněk MeSH
• regenerace nervu * MeSH
• tuková tkáň cytologie   metabolismus   MeSH
• Whartonův rosol cytologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

• MeSH
• autoimunita MeSH
• mezenchymální kmenové buňky MeSH
• transplantace kmenových buněk MeSH
• Publikační typ
• sborníky MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• hematologie a transfuzní lékařství
• biologie

The aim of this study was to compare the standard laboratory method of cultivation of mesenchymal multipotent stromal cells (MSC) and a novel technique of rapid MSC expansion focused on simple clinical use. MATERIAL AND METHODS Bone marrow mononuclear cells of donors were cultured for 14 days by the standard and the new cultivation method. The standard method (STD) was based on an alpha MEM medium supplemented with foetal calf serum (FCS). The new animal protein-free method (CLI) was based on the clinical grade medium CellgroTM, pooled human serum and human recombinant growth factors (EGF, PDGF-BB, M-CSF, FGF-2) supplemented with dexamethasone, insulin and ascorbic acid. The cell product was analyzed by flow cytometry. Furthermore, the cell products of STD and CLI methods were differentiated in vitro, and histochemical and immunohistochemical analyses, electron microscopy and elemental analysis were performed. Some cells were seeded on biodegradable scaffolds, in vivo implanted into immunodeficient mice for 6 weeks and evaluated by histological methods. RESULTS Yields of the CLI method after 14 days of cultivation were 40-fold higher than those obtained by the STD technique (p<0.05). Cell products of both STD and CLI methods fulfilled the criteria of MSC in terms of antigen expression assessed by flow cytometry, as well as osteogenic, chondrogenic and adipogenic in vitro differentiation assays. Moreover, these cells seeded on three-dimensional scaffolds cultured in osteogenic medium produced mineral deposits and a fibrillar extracellular matrix seen with the electron microscope. Deposits examined by element analysis contained calcium and phosphorus at a ratio of 5 to 3, which corresponded to hydroxyapatite. The cell product seeded on biodegradable scaffolds and implanted into immunodeficient mice was able to form a bone-like calcified tissue with blood supply of mouse origin. DISCUSSION The currently used methods of cultivation have certain disadvantages compared to the CLI technique, such as a longer cultivation period, need of primary expansion and reseeding and use of FCS with all its potential risks. High yields of cells obtained by the CLI method in a very short time make the use of cultured cells potentially suitable for an acute trauma management. Other therapeutic non-orthotopic applications of CLI-cultured cells have to be further investigated. CONCLUSIONS The CLI method is unique, rapid, simple and lacking the addition of animal proteins. CLI-cultured cells fulfil the criteria of MSC. The CLI method potentially allows for closed system cultivation in good manufacturing practice (GMP) conditions. It seems to be easily transferable to good clinical practice compared to other protocols and should extend the possibilities of cell therapy and tissue engineering of cartilage and bone. The new method is protected by Czech patent 301 148 and by europian patent EP 1999250 according to Czech and international laws.

• MeSH
• buněčná diferenciace MeSH
• dospělé kmenové buňky MeSH
• elektronová mikroskopie MeSH
• financování organizované MeSH
• histologie MeSH
• imunohistochemie MeSH
• interpretace statistických dat MeSH
• kultivační techniky metody   využití   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   MeSH
• myši MeSH
• průtoková cytometrie MeSH
• regenerativní lékařství MeSH
• růstový hormon MeSH
• statistika jako téma MeSH
• techniky in vitro MeSH
• tkáňové inženýrství metody   MeSH
• transplantace mezenchymálních kmenových buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH

BACKGROUND: A well-characterized method has not yet been established to reproducibly, efficiently, and safely isolate large numbers of clinical-grade multipotent human neural stem cells (hNSCs) from embryonic stem cells (hESCs). Consequently, the transplantation of neurogenic/gliogenic precursors into the CNS for the purpose of cell replacement or neuroprotection in humans with injury or disease has not achieved widespread testing and implementation. METHODS: Here, we establish an approach for the in vitro isolation of a highly expandable population of hNSCs using the manual selection of neural precursors based on their colony morphology (CoMo-NSC). The purity and NSC properties of established and extensively expanded CoMo-NSC were validated by expression of NSC markers (flow cytometry, mRNA sequencing), lack of pluripotent markers and by their tumorigenic/differentiation profile after in vivo spinal grafting in three different animal models, including (i) immunodeficient rats, (ii) immunosuppressed ALS rats (SOD1G93A), or (iii) spinally injured immunosuppressed minipigs. RESULTS: In vitro analysis of established CoMo-NSCs showed a consistent expression of NSC markers (Sox1, Sox2, Nestin, CD24) with lack of pluripotent markers (Nanog) and stable karyotype for more than 15 passages. Gene profiling and histology revealed that spinally grafted CoMo-NSCs differentiate into neurons, astrocytes, and oligodendrocytes over a 2-6-month period in vivo without forming neoplastic derivatives or abnormal structures. Moreover, transplanted CoMo-NSCs formed neurons with synaptic contacts and glia in a variety of host environments including immunodeficient rats, immunosuppressed ALS rats (SOD1G93A), or spinally injured minipigs, indicating these cells have favorable safety and differentiation characteristics. CONCLUSIONS: These data demonstrate that manually selected CoMo-NSCs represent a safe and expandable NSC population which can effectively be used in prospective human clinical cell replacement trials for the treatment of a variety of neurodegenerative disorders, including ALS, stroke, spinal traumatic, or spinal ischemic injury.

• MeSH
• buněčné linie MeSH
• lidé MeSH
• multipotentní kmenové buňky cytologie   MeSH
• nervové kmenové buňky cytologie   MeSH
• průtoková cytometrie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Využívání multipotentních mezenchymových stromálních buněk v rekonstrukční chirurgii má dlouhou historii. V současnosti se tyto buňky začínají využívat pro účely buněčné terapie dospělými kmenovými buňkami a tkáňového inženýrství. Výzkum dospěl i v ortopedii do preklinické fáze a vstupuje do klinických studií. Pro zajištění maximální bezpečnosti pro pacienta se použití těchto nových postupů musí řídit přísnými pravidly, která ještě nejsou celosvětově jednotná. Proto do doby, než budou v klinické praxi potvrzeny vědecké předpoklady, zůstane zlatým standardem pro zlepšení hojení kosti autologní štěp.

The application of multipotent mesenchymal stromal cells in reconstructive surgery has a long history. Currently these cells are used for purposes of somatic stem cell therapy and tissue engineering. Orthopedic basal research had also reached preclinical stage and entered clinical studies. For a patient safety assurance these new applications are strictly regulated but these regulations have not yet been harmonized worldwide. That is why autogenic bone graft stays a gold standard in bone regeneration until the experimental assumptions would be proven in clinical practice.

• Klíčová slova
• Fraxiparine,
• MeSH
• autologní transplantace metody   trendy   využití   MeSH
• buněčné kultury metody   normy   využití   MeSH
• lidé MeSH
• multipotentní kmenové buňky imunologie   transplantace   MeSH
• ortopedie metody   normy   trendy   MeSH
• regenerace kostí fyziologie   imunologie   MeSH
• tkáňové inženýrství metody   trendy   využití   MeSH
• transplantace mezenchymálních kmenových buněk metody   trendy   využití   MeSH
• transplantáty normy   trendy   využití   MeSH
• zákroky plastické chirurgie metody   trendy   využití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• směrnice pro lékařskou praxi MeSH

Jedním z nejspornějších problémů v současné biologii a medicíně je existence plasticity kmenových buněk. Termínem plasticita se míní fenotypový potenciál kmenových buněk, který je širší než jsou fenotypy diferencovaných buněk jejich původních tkání. Mnoho laboratoří již podalo důkaz o existenci plasticity kmenových buněk, ale vznikla celá řada námitek k prezentovaným výsledkům. Prezentujeme některé z námitek zpochybňujících údaje o plasticitě kmenových buněk. Tyto sporné otázky uvedeme do kontextu s tradičním pohledem na potenciál kmenových buněk a diverzifikaci buněčných fenotypu u dospělých a stárnoucích buněčných fenotypech. Vzhledem k malému prostoru se nebudeme zabývat plasticitou embryonálních a nádorových kmenových buněk. Naopak chceme v našem sdělení poukázat na některé otázky a problémy spojené s plasticitou kmenových buněk v dospělém organizmu a na úzký vztah k procesům regenerace a stárnutí. Z dosavadních experimentálních výsledků můžeme říci, že kmenové buňky pravděpodobně mají klíčovou úlohu v těchto procesech. Vzhledem k tomu, že se jedná o rychle se rozvíjející obor, je velmi obtížné najít jasné vysvědení pro nastolené otázky, a proto se omlouváme, že některé prezentované závěry potřebují další vysvědení a hlubší studium dané problematiky Toto sdělení představuje určitou sondu a úvod do diskuse určené celé řadě odborníků.

One of the most controversial problem in biology and medicine is the existence of stem ceUs plasticity. The term plasticity, as it is understood, is the potential of stem ceU fenotypes that is much broader that fenotypes of differentiated ceUs of their original tissues. Many laboratories have documented the existence of stem cell plasticity, however, there are still many objections to the reported results. Here, we present some of these objections to the reported results. Here, we present some of these objections questioning the data on stem cell plasticity in the context with the traditional view on stem cell potential and diversihcation in adult and aging cell fenotypes. As there is not enough space, we will not discuss the plasticity of embryonal and tumour stem ceUs. Instead, we wish to point out some problems associated with plasticity of stem ceUs in an adult organism and show their close relation to processes of regeneration and aging. Recent experimental results indicate that stem ceUs may have a key role in these processes. With regard to rapid developments in biology and medicine, some answers to the problems that seemed to be resolved according to the current state of knowledge, would have to be changed. This communication is just meant as an introduction and a probe for discussion for many concerned scientist.

• MeSH
• buněčná diferenciace MeSH
• finanční podpora výzkumu jako téma MeSH
• kmenové buňky fyziologie   MeSH
• mezenchymální kmenové buňky MeSH
• multipotentní kmenové buňky MeSH
• regenerace MeSH
• stárnutí MeSH
• totipotentní kmenové buňky MeSH

Clinical application of human multipotent mesenchymal stromal cells (hMSCs) requires their expansion to be safe and rapid. We aimed to develop an expansion protocol which would avoid xenogeneic proteins, including fetal calf serum (FCS), and which would shorten the cultivation time and avoid multiple passaging. First, we have compared research-grade alpha-MEM medium with clinical grade CellGro for Hematopoietic Cells' Medium. When FCS was used for supplementation and non-adherent cells were discarded, both media were comparable. Both media were comparable also when pooled human serum (hS) was used instead of FCS, but the numbers of hMSCs were lower when non-adherent cells were discarded. However, significantly more hMSCs were obtained both in alpha-MEM and in CellGro supplemented with hS when the non-adherent cells were left in the culture. Furthermore, addition of recombinant cytokines and other supplements (EGF, PDGF-BB, M-CSF, FGF-2, dexamethasone, insulin and ascorbic acid) to the CellGro co-culture system with hS led to 40-fold increase of hMSCs' yield after two weeks of cultivation compared to alpha-MEM with FCS. The hMSCs expanded in the described co-culture system retain their osteogenic, adipogenic and chondrogenic differentiation potential in vitro and produce bone-like mineralized tissue when propagated on 3D polylactide scaffolds in immunodeficient mice. Our protocol thus allows for very effective one-step, xenogeneic protein-free expansion of hMSCs, which can be easily transferred into good manufacturing practice (GMP) conditions for large-scale, clinical-grade production of hMSCs for purposes of tissue engineering.

• MeSH
• buněčná diferenciace fyziologie   MeSH
• buněčné kultury MeSH
• buňky stromatu cytologie   fyziologie   MeSH
• cytokiny metabolismus   MeSH
• dospělí MeSH
• kosti a kostní tkáň fyziologie   MeSH
• kultivační média chemie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   fyziologie   MeSH
• mezibuněčné signální peptidy a proteiny metabolismus   MeSH
• mladý dospělý MeSH
• multipotentní kmenové buňky cytologie   fyziologie   MeSH
• myši inbrední NOD MeSH
• myši MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tkáňové inženýrství metody   MeSH
• transplantace mezenchymálních kmenových buněk MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• myši MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• hodnotící studie MeSH
• práce podpořená grantem MeSH