Standardized xeno- and serum-free culture platform enables large-scale expansion of high-quality mesenchymal stem/stromal cells from perinatal and adult tissue sources

Background aimsMesenchymal stem/stromal cells (MSCs) are of interest for the treatment of graft-versus-host disease, autoimmune diseases, osteoarthritis and neurological and cardiovascular diseases. Increasing numbers of clinical trials emphasize the need for standardized manufacturing of these cells. However, many challenges related to diverse isolation and expansion protocols and differences in cell tissue sources exist. As a result, the cell products used in numerous trials vary greatly in characteristics and potency.MethodsThe authors have established a standardized culture platform using xeno- and serum-free commercial media for expansion of MSCs derived from umbilical cord (UC), bone marrow and adipose-derived (AD) and examined their functional characteristics.ResultsMSCs from the tested sources stably expanded in vitro and retained their biomarker expression and normal karyotype at early and later passages and after cryopreservation. MSCs were capable of colony formation and successfully differentiated into osteogenic, adipogenic and chondrogenic lineages. Pilot expansion of UC-MSCs and AD-MSCs to clinical scale revealed that the cells met the required quality standard for therapeutic applications.ConclusionsThe authors’ data suggest that xeno- and serum-free culture conditions are suitable for large-scale expansion and enable comparative study of MSCs of different origins. This is of importance for therapeutic purposes, especially because of the numerous variations in pre-clinical and clinical protocols for MSC-based products.     

Umbilical cord–derived mesenchymal stromal cells in cardiovascular disease: review of preclinical and clinical data

The human umbilical cord has recently emerged as an attractive potential source of mesenchymal stromal cells (MSCs) to be adopted for use in regenerative medicine. Umbilical cord MSCs (UC-MSCs) not only share the same features of all MSCs such as multi-lineage differentiation, paracrine functions and immunomodulatory properties, they also have additional advantages, such as no need for bone marrow aspiration and higher self-renewal capacities. They can be isolated from various compartments of the umbilical cord (UC) and can be used for autologous or allogeneic purposes. In the past decade, they have been adopted in cardiovascular disease and have shown promising results mainly due to their pro-angiogenic and anti-inflammatory properties. This review offers an overview of the biological properties of UC-MSCs describing available pre-clinical and clinical data with respect to their potential therapeutic use in cardiovascular regeneration, with current challenges and future directions discussed.     

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.     

Wharton’s jelly‐derived stromal cells and their cell therapy applications in allogeneic haematopoietic stem cell transplantation

For decades, mesenchymal stromal cells (MSCs) have been of great interest in the fields of regenerative medicine, tissue engineering and immunomodulation. Their tremendous potential makes it desirable to cryopreserve and bank MSCs to increase their accessibility and availability. Postnatally derived MSCs seem to be of particular interest because they are harvested after delivery without ethical controversy, they have the capacity to expand at a higher rate than adult‐derived MSCs, in which expansion decreases with ageing, and they have demonstrated immunological and haematological supportive properties similar to those of adult‐derived MSCs. In this review, we focus on MSCs obtained from Wharton''s jelly (the mucous connective tissue of the umbilical cord between the amniotic epithelium and the umbilical vessels). Wharton''s jelly MSCs (WJ‐MSCs) are a good candidate for cellular therapy in haematology, with accumulating data supporting their potential to sustain haematopoietic stem cell engraftment and to modulate alloreactivity such as Graft Versus Host Disease (GVHD). We first present an overview of their in‐vitro properties and the results of preclinical murine models confirming the suitability of WJ‐MSCs for cellular therapy in haematology. Next, we focus on clinical trials and discuss tolerance, efficacy and infusion protocols reported in haematology for GVHD and engraftment.     

Differential reduction of reactive oxygen species by human tissue-specific mesenchymal stem cells from different donors under oxidative stress

Clinical trials using human Mesenchymal Stem Cells (MSCs) have shown promising results in the treatment of various diseases. Different tissue sources, such as bone marrow, adipose tissue, dental pulp and umbilical cord, are being routinely used in regenerative medicine. MSCs are known to reduce increased oxidative stress levels in pathophysiological conditions. Differences in the ability of MSCs from different donors and tissues to ameliorate oxidative damage have not been reported yet. In this study, for the first time, we investigated the differences in the reactive oxygen species (ROS) reduction abilities of tissue-specific MSCs to mitigate cellular damage in oxidative stress. Hepatic Stellate cells (LX-2) and cardiomyocytes were treated with Antimycin A (AMA) to induce oxidative stress and tissue specific MSCs were co-cultured to study the reduction in ROS levels. We found that both donor’s age and source of tissue affected the ability of MSCs to reduce increased ROS levels in damaged cells. In addition, the abilities of same MSCs differed in LX-2 and cardiomyocytes in terms of magnitude of reduction of ROS, suggesting that the type of recipient cells should be kept in consideration when using MSCs in regenerative medicine for treatment purposes.     

Immunosuppressive properties of mesenchymal stem cells: advances and applications

Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, such as bone marrow, skeletal muscle, dental pulp, bone, umbilical cord and adipose tissue. MSCs are used in regenerative medicine mainly based on their capacity to differentiate into specific cell types and also as bioreactors of soluble factors that will promote tissue regeneration from the damaged tissue cellular progenitors. In addition to these regenerative properties, MSCs hold an immunoregulatory capacity, and elicit immunosuppressive effects in a number of situations. Not only are they immunoprivileged cells, due to the low expression of class II Major Histocompatibilty Complex (MHC-II) and costimulatory molecules in their cell surface, but they also interfere with different pathways of the immune response by means of direct cell-to-cell interactions and soluble factor secretion. In vitro, MSCs inhibit cell proliferation of T cells, B-cells, natural killer cells (NK) and dendritic cells (DC), producing what is known as division arrest anergy. Moreover, MSCs can stop a variety of immune cell functions: cytokine secretion and cytotoxicity of T and NK cells; B cell maturation and antibody secretion; DC maturation and activation; as well as antigen presentation. It is thought that MSCs need to be activated to exert their immunomodulation skills. In this scenario, an inflammatory environment seems to be necessary to promote their effect and some inflammation-related molecules such as tumor necrosis factor-α and interferon-γ might be implicated. It has been observed that MSCs recruit T-regulatory lymphocytes (Tregs) to both lymphoid organs and graft. There is great controversy concerning the mechanisms and molecules involved in the immunosuppressive effect of MSCs. Prostaglandin E2, transforming growth factor-β, interleukins- 6 and 10, human leukocyte antigen-G5, matrix metalloproteinases, indoleamine-2,3-dioxygenase and nitric oxide are all candidates under investigation. In vivo studies have shown many discrepancies regarding the immunomodulatory properties of MSCs. These studies have been designed to test the efficacy of MSC therapy in two different immune settings: the prevention or treatment of allograft rejection episodes, and the ability to suppress abnormal immune response in autoimmune and inflammatory diseases. Preclinical studies have been conducted in rodents, rabbits and baboon monkeys among others for bone marrow, skin, heart, and corneal transplantation, graft versus host disease, hepatic and renal failure, lung injury, multiple sclerosis, rheumatoid arthritis, diabetes and lupus diseases. Preliminary results from some of these studies have led to human clinical trials that are currently being carried out. These include treatment of autoimmune diseases such as Crohn's disease, ulcerative colitis, multiple sclerosis and type 1 diabetes mellitus; prevention of allograft rejection and enhancement of the survival of bone marrow and kidney grafts; and treatment of resistant graft versus host disease. We will try to shed light on all these studies, and analyze why the results are so contradictory.     

Treatment of graft-versus-host-disease with mesenchymal stromal cells

Mesenchymal stromal cells (MSC) are a population of phenotypically heterogeneous cells that can be isolated from many readily accessible tissues, including bone marrow, umbilical cord, placenta and adipose tissue, where they form part of the supportive, stromal micro-environment. Extensive ex vivo and pre-clinical data suggest that subpopulations within MSC contribute to immunomodulation of the host, without provoking immunologic responses from alloreactive T cells or other effector cells, as well as contributing to tissue repair. These unique properties make MSC an ideal investigational agent for treating graft-versus-host disease (GvHD). Therapeutic trials with varied MSC dosing schedules and clinical end-points have shown mixed results. We have reviewed the biology of MSC gleaned from pre-clinical models, and summarized the results of clinical trials utilizing MSC for the treatment of acute and chronic GvHD.     

Differentiation of Human Umbilical Cord Matrix Mesenchymal Stem Cells into Neural-Like Progenitor Cells and Maturation into an Oligodendroglial-Like Lineage

Mesenchymal stem cells (MSCs) are viewed as safe, readily available and promising adult stem cells, which are currently used in several clinical trials. Additionally, their soluble-factor secretion and multi-lineage differentiation capacities place MSCs in the forefront of stem cell types with expected near-future clinical applications. In the present work MSCs were isolated from the umbilical cord matrix (Wharton''s jelly) of human umbilical cord samples. The cells were thoroughly characterized and confirmed as bona-fide MSCs, presenting in vitro low generation time, high proliferative and colony-forming unit-fibroblast (CFU-F) capacity, typical MSC immunophenotype and osteogenic, chondrogenic and adipogenic differentiation capacity. The cells were additionally subjected to an oligodendroglial-oriented step-wise differentiation protocol in order to test their neural- and oligodendroglial-like differentiation capacity. The results confirmed the neural-like plasticity of MSCs, and suggested that the cells presented an oligodendroglial-like phenotype throughout the differentiation protocol, in several aspects sharing characteristics common to those of bona-fide oligodendrocyte precursor cells and differentiated oligodendrocytes.     

人脐带和胎盘不同层次间充质干细胞的生物学特性分析

间充质干细胞(mesenchymal stem cell,MSC)在再生医学中具有广阔的应用前景,其临床转化应用已成为研究热点,如何大量获取原代间充质干细胞以及针对不同疾病选择最为合适的细胞来源是关键.为了探讨不同来源间充质干细胞的异同,为临床治疗与研究选择合适的种子细胞提供参考,文中比较了人脐带和胎盘不同层次间充质干...     

Current understanding of mesenchymal stem cells in liver diseases

Liver diseases caused by various factors have become a significant threat to public health worldwide. Liver transplantation has been considered as the only effective treatment for end-stage liver diseases; however, it is limited by the shortage of donor organs, postoperative complications, long-term immunosuppression, and high cost of treatment. Thus, it is not available for all patients. Recently, mesenchymal stem cells (MSCs) transplantation has been extensively explored for repairing hepatic injury in various liver diseases. MSCs are multipotent adult progenitor cells originated from the embryonic mesoderm, and can be found in mesenchymal tissues including the bone marrow, umbilical cord blood, adipose tissue, liver, lung, and others. Although the precise mechanisms of MSC transplantation remain mysterious, MSCs have been demonstrated to be able to prevent the progression of liver injury and improve liver function. MSCs can self-renew by dividing, migrating to injury sites and differentiating into multiple cell types including hepatocytes. Additionally, MSCs have immune-modulatory properties and release paracrine soluble factors. Indeed, the safety and effectiveness of MSC therapy for liver diseases have been demonstrated in animals. However, pre-clinical and clinical trials are largely required to confirm its safety and efficacy before large scale clinical application. In this review, we will explore the molecular mechanisms underlying therapeutic effects of MSCs on liver diseases. We also summarize clinical advances in MSC-based therapies.     

Exosomes derived from uMSCs promote hair regrowth in alopecia areata through accelerating human hair follicular keratinocyte proliferation and migration

Alopecia areata (AA) is a complex genetic disease that results in hair loss due to an autoimmune-mediated attack on the hair follicle. Mesenchymal stem cells (MSCs) have great potential to induce hair regeneration due to their strong secretion ability and multidirectional differentiation. Recent studies have revealed that the therapeutic potential of MSCs comes from their secretion ability, which can produce large amounts of bioactive substances and regulate the key physiological functions of subjects. The secretion products of MSCs, such as vesicles, exosomes, and conditioned media, have significant advantages in preparing of biological products derived from stem cells. Human umbilical cord mesenchymal stem cells (uMSCs) are the best choice for exosome production. uMSCs are obtained from the human umbilical cord. The umbilical cord is easy to obtain, and the efficiency of uMSCs isolation and culture higher than that of obtaining MSCs from bone marrow or adipose tissue. In this study, we investigated the effects of exosomes released from uMSCs in AA mice. In summary, due to easy isolation and cultivation, simple preparation, and convenient storage, it is possible to obtain uMSCs, or uMSCs exosomes for research and clinical treatment.     

Application of mesenchymal stem cells in regenerative medicine: A new approach in modern medical science

Mesenchymal Stem Cells (MSCs) are non-hematopoietic and multipotent stem cells, which have been considered in regenerative medicine. These cells are easily separated from different sources, such as bone marrow (BM), umbilical cord (UC), adipose tissue (AT), and etc. MSCs have the differentiation capability into chondrocytes, osteocytes, and adipocytes; This differentiation potential along with the paracrine properties have made them a key choice for tissue repair. MSCs also have various advantages over other stem cells, which is why they have been extensively studied in recent years. The effectiveness of MSCs-based therapies depend on several factors, including differentiation status at the time of use, concentration per injection, delivery method, the used vehicle, and the nature and extent of the damage. Although, MSCs have emerged promising sources for regenerative medicine, there are potential risks regarding their safety in their clinical use, including tumorigenesis, lack of availability, aging, and sensitivity to toxic environments. In this study, we aimed to discuss how MSCs may be useful in treating defects and diseases. To this aim, we will review recent advances of MSCs action mechanisms in regenerative medicine, as well as the most recent clinical trials. We will also have a brief overview of MSCs resources, differences between their sources, culture conditions, extraction methods, and clinical application of MSCs in various fields of regenerative medicine.     

间充质干细胞分化为内皮细胞的意义和细胞学基础

间充质干细胞(mesenchymalstemcells,MSCs)主要存在于骨髓中,是多潜能干细胞,在脐血、外周血、脂肪、皮肤等多种组织中也相继分离出MSCs。MSCs具有独特的免疫特性,在异种异体环境内长期存在,使其临床应用前景更为广泛。目前,MSCs的分离培养、诱导分化及鉴定体系已趋成熟,理论上可分化为所有中胚层来源的细胞,内皮细胞来源于中胚层,因此MSCs具有分化为内皮细胞的可能性。本文对MSCs内皮分化意义和细胞学基础及其新近的研究进展作一综述。     

Characterization of mesenchymal stem cells isolated from the human umbilical cord

Numerous papers have reported that mesenchymal stem cells (MSCs) can be isolated from various sources such as bone marrow, adipose tissue and others. Nonetheless it is an open question whether MSCs isolated from different sources represent a single cell lineage or if cells residing in different organs are separate members of a family of MSCs. Subendothelial tissue of the umbilical cord vein has been shown to be a promising source of MSCs. The aim of this study was to isolate and characterize cells derived from the subendothelial layer of umbilical cord veins as regards their clonogenicity and differentiation potential. The results from these experiments show that cells isolated from the umbilical cord vein displayed fibroblast-like morphology and grew into colonies. Immunophenotyping by flow cytometry revealed that the isolated cells were negative for the hematopoietic line markers HLA-DR and CD34 but were positive for CD29, CD90 and CD73. The isolated cells were also positive for survivin, Bcl-2, vimentin and endoglin, as confirmed by RT-PCR and immunofluorescence. These cells can be induced to differentiate into osteogenic and adipogenic cells, but a new finding is that these cells can be induced to differentiate into endothelial cells expressing CD31, vWF and KDR-2, and also form vessel-like structures in Matrigel. The differentiated cells stopped expressing survivin, thus showing a diminished proliferative potential. It can be assumed that the subendothelial layer of the umbilical cord vein contains a population of cells with the overall characteristics of MSCs, with the additional capability to transform into endothelial cells.     

Transplantation of human villous trophoblasts preserves cardiac function in mice with acute myocardial infarction

Over the past decade, cell therapies have provided promising strategies for the treatment of ischaemic cardiomyopathy. Particularly, the beneficial effects of stem cells, including bone marrow stem cells (BMSCs), endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), have been demonstrated by substantial preclinical and clinical studies. Nevertheless stem cell therapy is not always safe and effective. Hence, there is an urgent need for alternative sources of cells to promote cardiac regeneration. Human villous trophoblasts (HVTs) play key roles in embryonic implantation and placentation. In this study, we show that HVTs can promote tube formation of human umbilical vein endothelial cells (HUVECs) on Matrigel and enhance the resistance of neonatal rat cardiomyocytes (NRCMs) to oxidative stress in vitro. Delivery of HVTs to ischaemic area of heart preserved cardiac function and reduced fibrosis in a mouse model of acute myocardial infarction (AMI). Histological analysis revealed that transplantation of HVTs promoted angiogenesis in AMI mouse hearts. In addition, our data indicate that HVTs exert their therapeutic benefit through paracrine mechanisms. Meanwhile, injection of HVTs to mouse hearts did not elicit severe immune response. Taken together, our study demonstrates HVT may be used as a source for cell therapy or a tool to study cell‐derived soluble factors for AMI treatment.     

Mesenchymal stem cells: biology and clinical potential in type 1 diabetes therapy

Mesenchymal stem cells (MSCs) can be derived from adult bone marrow, fat and several foetal tissues. In vitro , MSCs have the capacity to differentiate into multiple mesodermal and non-mesodermal cell lineages. Besides, MSCs possess immunosuppressive effects by modulating the immune function of the major cell populations involved in alloantigen recognition and elimination. The intriguing biology of MSCs makes them strong candidates for cell-based therapy against various human diseases. Type 1 diabetes is caused by a cell-mediated autoimmune destruction of pancreatic β-cells. While insulin replacement remains the cornerstone treatment for type 1 diabetes, the transplantation of pancreatic islets of Langerhans provides a cure for this disorder. And yet, islet transplantation is limited by the lack of donor pancreas. Generation of insulin-producing cells (IPCs) from MSCs represents an attractive alternative. On the one hand, MSCs from pancreas, bone marrow, adipose tissue, umbilical cord blood and cord tissue have the potential to differentiate into IPCs by genetic modification and/or defined culture conditions In vitro . On the other hand, MSCs are able to serve as a cellular vehicle for the expression of human insulin gene. Moreover, protein transduction technology could offer a novel approach for generating IPCs from stem cells including MSCs. In this review, we first summarize the current knowledge on the biological characterization of MSCs. Next, we consider MSCs as surrogate β-cell source for islet transplantation, and present some basic requirements for these replacement cells. Finally, MSCs-mediated therapeutic neovascularization in type 1 diabetes is discussed.     

Intracellular immunoglobulins in Namalva and U266 cells cocultivated with mesenchymal stromal cells

The data concerning the influence of mesenchymal stromal cells (MSCs) on immunoglobulin (Ig) production are contradictory. Most results were obtained using MSC derived from bone marrow. The properties of MSCs obtained from other tissues are not well studied. In the present work, MSC cultures have been established from umbilical cord, adipose tissue, and bone marrow of healthy donors, as well as from bone marrow of patients with autoimmune diseases. MSCs from all these sources exhibited similar surface markers. We assayed the influence of MSC cocultivation at exponential or stationary growth phases on IgM content in Namalva and IgE content in U266 cells. Bone marrow MSCs from healthy donors did not affect IgM and IgE production. Proliferating MSCs from patients with Crohn’s disease and multiple sclerosis stimulated Ig production. Exponentially growing MSCs derived from umbilical cord and adipose tissue also stimulated Ig synthesis. MSCs at stationary cultures enhanced IgM production in Namalva (cells) and suppressed IgE synthesis in U266 cells. Thus, MSCs from various tissues with common phenotypes differed in their capacity to modulate Ig production by B-lymphoid cells. The effect of MSCs depends on their growth stage and may be different for lymphoblastoid and myeloma cells.     

Characterizing human mesenchymal stromal cells’ immune-modulatory potency using targeted lipidomic profiling of sphingolipids

Cell therapies are expected to increase over the next decade owing to increasing demand for clinical applications. Mesenchymal stromal cells (MSCs) have been explored to treat a number of diseases, with some successes in early clinical trials. Despite early successes, poor MSC characterization results in lessened therapeutic capacity once in vivo. Here, we characterized MSCs derived from bone marrow (BM), adipose tissue and umbilical cord tissue for sphingolipids (SLs), a class of bioactive lipids, using liquid chromatography/tandem mass spectrometry. We found that ceramide levels differed based on the donor's sex in BM-MSCs. We detected fatty acyl chain variants in MSCs from all three sources. Linear discriminant analysis revealed that MSCs separated based on tissue source. Principal component analysis showed that interferon-γ–primed and unstimulated MSCs separated according to their SL signature. Lastly, we detected higher ceramide levels in low indoleamine 2,3-dioxygenase MSCs, indicating that sphingomyelinase or ceramidase enzymatic activity may be involved in their immune potency.     

生长因子诱导人脐带/胎盘间充质干细胞定向分化

近年来,间充质干细胞(mesenchymal stem cell,MSC)已成为干细胞领域的研究热点,其不仅支持造血系统,还可在特定的培养条件下向多种组织细胞分化。人脐带和胎盘来源的MSC取材容易,较骨髓间充质干细胞有更广泛的应用前景。本文就含有特定生长因子的培养基诱导人脐带MSC和人胎盘MSC定向分化的研究进展作一简要的综述。