Advances in ex vivo expansion of hematopoietic stem and progenitor cells for clinical applications.

As caretakers of the hematopoietic system, hematopoietic stem cells assure a lifelong supply of differentiated populations that are responsible for critical bodily functions, including oxygen transport, immunological protection and coagulation. Due to the far-reaching influence of the hematopoietic system, hematological disorders typically have a significant impact on the lives of individuals, even becoming fatal. Hematopoietic cell transplantation was the first effective therapeutic avenue to treat such hematological diseases. Since then, key use and manipulation of hematopoietic stem cells for treatments has been aspired to fully take advantage of such an important cell population. Limited knowledge on hematopoietic stem cell behavior has motivated in-depth research into their biology. Efforts were able to uncover their native environment and characteristics during development and adult stages. Several signaling pathways at a cellular level have been mapped, providing insight into their machinery. Important dynamics of hematopoietic stem cell maintenance were begun to be understood with improved comprehension of their metabolism and progressive aging. These advances have provided a solid platform for the development of innovative strategies for the manipulation of hematopoietic stem cells. Specifically, expansion of the hematopoietic stem cell pool has triggered immense interest, gaining momentum. A wide range of approaches have sprouted, leading to a variety of expansion systems, from simpler small molecule-based strategies to complex biomimetic scaffolds. The recent approval of Omisirge, the first expanded hematopoietic stem and progenitor cell product, whose expansion platform is one of the earliest, is predictive of further successes that might arise soon. In order to guarantee the quality of these ex vivo manipulated cells, robust assays that measure cell function or potency need to be developed. Whether targeting hematopoietic engraftment, immunological differentiation potential or malignancy clearance, hematopoietic stem cells and their derivatives need efficient scaling of their therapeutic potency. In this review, we comprehensively view hematopoietic stem cells as therapeutic assets, going from fundamental to translational.

Dual production of human mesenchymal stromal cells and derived extracellular vesicles in a dissolvable microcarrier-based stirred culture system.

BACKGROUND & AIMS: Cell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, we aimed at establishing a scalable culture system to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) and their derived extracellular vesicles (EVs) by using dissolvable microcarriers combined with xeno(geneic)-free culture medium. METHODS: MSC(WJ) isolated from three donors were cultured at a starting density of 1 × 106 cells per spinner flask, i.e., 2.8 × 103 cells per cm2 of dissolvable microcarrier surface area. After a 6-day expansion period of MSC(WJ), extracellular vesicles (EVs) were produced for 24 h. RESULTS: Taking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24 h) and achieved 15.8 ± 0.7-fold expansion after 6 days of culture. Notably, dissolution of the microcarriers was achieved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype and by multilineage differentiation assays. Considering the growing interest in MSC-derived EVs, which are known to be mediators of the therapeutic features of MSC, this platform also was evaluated for EV production. Upon a 24-h period of conditioning, secreted EVs were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6 ± 30.2 nm) and expressed EV markers (CD63, CD9 and syntenin-1). CONCLUSIONS: Taken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.

Natural killer cells suppress human cutaneous squamous cell carcinoma cancer cell survival and tumor growth.

Cutaneous squamous cell carcinoma (CSCC), which develops in response to ultraviolet irradiation exposure, is among the most common cancers. CSCC lesions can be removed by surgical excision, but 4.5% of these cancers reappear as aggressive and therapy-resistant tumors. CSCC tumors display a high mutation burden, and tumor frequency is dramatically increased in immune-suppressed patients, indicating a vital role for the immune system in controlling cancer development. Natural killer cells (NK cells) play a key role in cancer immune surveillance, and recent studies suggest that NK cells from healthy donors can be expanded from peripheral blood for use in therapy. In the present study, we test the ability of ex vivo expanded human NK cells to suppress the CSCC cell cancer phenotype and reduce tumor growth. We expanded human NK cells from multiple healthy donors, in the presence of IL-2, and tested their ability to suppress the CSCC cell cancer phenotype. NK cell treatment produced a dose-dependent reduction in SCC-13 and HaCaT cell spheroid growth and matrigel invasion and induced SCC-13 and HaCaT cell apoptosis as evidenced by increased procaspase 9, procaspase 3, and PARP cleavage. Moreover, two important CSCC cell pro-cancer signaling pathways, YAP1/TAZ/TEAD and MEK1/2-ERK1/2, were markedly reduced. Furthermore, tail-vein injection of NK cells markedly suppressed the growth of SCC-13 xenograft tumors in NSG mice, which was also associated with a reduction in YAP1 and MEK1/2-P levels and enhanced apoptosis. These findings show that NK cell treatment suppresses CSCC cell spheroid formation, invasion, viability, and tumor growth, suggesting NK cell treatment may be a candidate therapy for CSCC.

Current Perspectives on "Off-The-Shelf" Allogeneic NK and CAR-NK Cell Therapies.

Natural killer cells (NK cells) are the first line of the innate immune defense system, primarily located in peripheral circulation and lymphoid tissues. They kill virally infected and malignant cells through a balancing play of inhibitory and stimulatory receptors. In pre-clinical investigational studies, NK cells show promising anti-tumor effects and are used in adoptive transfer of activated and expanded cells, ex-vivo. NK cells express co-stimulatory molecules that are attractive targets for the immunotherapy of cancers. Recent clinical trials are investigating the use of CAR-NK for different cancers to determine the efficiency. Herein, we review NK cell therapy approaches (NK cell preparation from tissue sources, ways of expansion ex-vivo for "off-the-shelf" allogeneic cell-doses for therapies, and how different vector delivery systems are used to engineer NK cells with CARs) for cancer immunotherapy.

Successful Derivation of an Induced Pluripotent Stem Cell Line from a Genetically Nonpermissive Enhanced Green Fluorescent Protein-Transgenic FVB/N Mouse Strain.

The embryonic stem cell line derivation from nonpermissive mouse strains is a challenging and highly inefficient process. The cellular reprogramming strategy provides an alternative route for generating pluripotent stem cell (PSC) lines from such strains. In this study, we successfully derived an enhanced green fluorescent protein (EGFP)-transgenic "N9" induced pluripotent stem cell (iPS cell, iPSC) line from the FVB/N strain-derived mouse embryonic fibroblasts (MEFs). The exposure of MEFs to human OCT4, SOX2, KLF4, and c-MYC (OSKM) transgenes via lentiviral transduction resulted in complete reprogramming. The N9 iPS cell line demonstrated all the criteria of a typical mouse PSC line, including normal colony morphology and karyotype (40,XY), high replication and propagation efficiencies, expression of the pluripotency-associated genes, spontaneous differentiation to three germ lineage-derived cell types, and robust potential of chimeric blastocyst formation. Taken together, using human OSKM genes for transduction, we report, for the first time, the successful derivation of an EGFP-expressing iPS cell line from a genetically nonpermissive transgenic FVB/N mouse. This cell line could provide opportunities for designing protocols for efficient derivation of PSC lines from other nonpermissive strains and developing mouse models of human diseases.

CAR T Cell Therapy: A Game Changer in Cancer Treatment.

The development of novel targeted therapies with acceptable safety profiles is critical to successful cancer outcomes with better survival rates. Immunotherapy offers promising opportunities with the potential to induce sustained remissions in patients with refractory disease. Recent dramatic clinical responses in trials with gene modified T cells expressing chimeric antigen receptors (CARs) in B-cell malignancies have generated great enthusiasm. This therapy might pave the way for a potential paradigm shift in the way we treat refractory or relapsed cancers. CARs are genetically engineered receptors that combine the specific binding domains from a tumor targeting antibody with T cell signaling domains to allow specifically targeted antibody redirected T cell activation. Despite current successes in hematological cancers, we are only in the beginning of exploring the powerful potential of CAR redirected T cells in the control and elimination of resistant, metastatic, or recurrent nonhematological cancers. This review discusses the application of the CAR T cell therapy, its challenges, and strategies for successful clinical and commercial translation.

Research using Mesenchymal Stem/Stromal Cells: quality metric towards developing a reference material.

Mesenchymal stem/stromal cells (MSCs) have been extensively investigated for their regenerative, immune-modulatory, and wound healing properties. While the laboratory studies have suggested that MSC's have a unique potential for modulating the etiopathology of multiple diseases, the results from clinical trials have not been encouraging or reproducible. One of the explanations for such variability is explained by the "art" of isolating and propagating MSCs. Therefore, establishing more than minimal criteria to define MSC would help understand best protocols to isolate, propagate and deliver MSCs. Developing a calibration standard, a database and a set of functional tests would be a better quality metric for MSCs. In this review, we discuss the importance of selecting a standard, issues associated with coming up with such a standard and how these issues can be mitigated.

Extracellular vesicles from bone marrow mesenchymal stem/stromal cells transport tumor regulatory microRNA, proteins, and metabolites.

Human mesenchymal stem/stromal cells (hMSCs) have been shown to support breast cancer cell proliferation and metastasis, partly through their secretome. hMSCs have a remarkable ability to survive for long periods under stress, and their secretome is tumor supportive. In this study, we have characterized the cargo of extracellular vesicular (EV) fraction (that is in the size range of 40-150nm) of serum deprived hMSCs (SD-MSCs). Next Generation Sequencing assays were used to identify small RNA secreted in the EVs, which indicated presence of tumor supportive miRNA. Further assays demonstrated the role of miRNA-21 and 34a as tumor supportive miRNAs. Next, proteomic assays revealed the presence of ≈150 different proteins, most of which are known tumor supportive factors such as PDGFR-ß, TIMP-1, and TIMP-2. Lipidomic assays verified presence of bioactive lipids such as sphingomyelin. Furthermore, metabolite assays identified the presence of lactic acid and glutamic acid in EVs. The co-injection xenograft assays using MCF-7 breast cancer cells demonstrated the tumor supportive function of these EVs. To our knowledge this is the first comprehensive -omics based study that characterized the complex cargo of extracellular vesicles secreted by hMSCs and their role in supporting breast cancers.

A xenogeneic-free bioreactor system for the clinical-scale expansion of human mesenchymal stem/stromal cells.

The large cell doses (>1 × 10(6) cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex-vivo expansion is critical to provide a safe and reliable cell product. Bioprocess engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost-effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)-free microcarrier-based culture system was successfully established for bone marrow (BM) MSC and adipose tissue-derived stem/stromal cell (ASC) cultivation using a 1L-scale controlled stirred-tank bioreactor, allowing the production of (1.1 ± 0.1) × 10(8) and (4.5 ± 0.2) × 10(7) cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat ) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat . Also, the three different feeding regimes studied-(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed-batch addition of concentrated nutrients and growth factors every 2 days-yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA-DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier-based stirred culture system under xeno-free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP-compliant clinical-scale MSC production system.

Identification of a population of epidermal squamous cell carcinoma cells with enhanced potential for tumor formation.

Epidermal squamous cell carcinoma is among the most common cancers in humans. These tumors are comprised of phenotypically diverse populations of cells that display varying potential for proliferation and differentiation. An important goal is identifying cells from this population that drive tumor formation. To enrich for tumor-forming cells, cancer cells were grown as spheroids in non-attached conditions. We show that spheroid-selected cells form faster growing and larger tumors in immune-compromised mice as compared to non-selected cells. Moreover, spheroid-selected cells gave rise to tumors following injection of as few as one hundred cells, suggesting these cells have enhanced tumor-forming potential. Cells isolated from spheroid-selected tumors retain an enhanced ability to grow as spheroids when grown in non-attached culture conditions. Thus, these tumor-forming cells retain their phenotype following in vivo passage as tumors. Detailed analysis reveals that spheroid-selected cultures are highly enriched for expression of epidermal stem cell and embryonic stem cell markers, including aldehyde dehydrogenase 1, keratin 15, CD200, keratin 19, Oct4, Bmi-1, Ezh2 and trimethylated histone H3. These studies indicate that a subpopulation of cells that possess stem cell-like properties and express stem cell markers can be derived from human epidermal cancer cells and that these cells display enhanced ability to drive tumor formation.

Development of fully defined xeno-free culture system for the preparation and propagation of cell therapy-compliant human adipose stem cells.

INTRODUCTION: Adipose tissue is an attractive and abundant source of multipotent stem cells. Human adipose stem cells (ASCs) have shown to have therapeutic relevancy in diverse clinical applications. Nevertheless, expansion of ASCs is often necessary before performing clinical studies. Standard in vitro cell-culture techniques use animal-derived reagents that should be avoided in clinical use because of safety issues. Therefore, xeno- and serum-free (XF/SF) reagents are highly desirable for enhancing the safety and quality of the transplanted ASCs. METHODS: In the current study, animal component-free isolation and cell-expansion protocols were developed for ASCs. StemPro MSC SFM XF medium with either CELLstart™ CTS™ coating or Coating Matrix Kit were tested for their ability to support XF/SF growth. Basic stem-cell characteristics such as immunophenotype (CD3, CD11a, CD14, CD19, CD34, CD45RO, CD54, CD73, CD80, CD86, CD90, CD105, HLA-DR), proliferation, and differentiation potential were assessed in XF/SF conditions and compared with human serum (HS) or traditionally used fetal bovine serum (FBS) cultures. RESULTS: ASCs cultured in XF/SF conditions had significantly higher proliferation rates compared with HS/FBS cultures. Characteristic immunophenotypes of ASCs were maintained in every condition; however, cells expanded in XF/SF conditions showed significantly lower expression of CD54 (intercellular adhesion molecule 1, ICAM-1) at low passage number. Further, multilineage differentiation potential of ASCs was maintained in every culture condition. CONCLUSIONS: Our findings demonstrated that the novel XF/SF conditions maintained the basic stem cell features of ASCs and the animal-free workflow followed in this study has great potential in clinical cell therapies.

Biochemistry of epidermal stem cells.

BACKGROUND: The epidermis is an important protective barrier that is essential for maintenance of life. Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processes must be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locations in the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment of this tissue. SCOPE OF REVIEW: A great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifying stem cell niche locations, and in understanding how stem cell populations are related. We discuss these studies as they apply to understanding normal epidermal homeostasis and skin cancer. MAJOR CONCLUSIONS: An assortment of stem cell markers have been identified that permit assignment of stem cells to specific regions of the epidermis, and progress has been made in understanding the role of these cells in normal epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations exist in epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair in damaged epidermis. GENERAL SIGNIFICANCE: Understanding epidermal stem cell biology is likely to lead to important therapies for treating skin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Development and characterization of a clinically compliant xeno-free culture medium in good manufacturing practice for human multipotent mesenchymal stem cells.

Human multipotent mesenchymal stem cell (MSC) therapies are currently being tested in clinical trials for Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, cartilage damage, and cardiac diseases. Despite remarkable progress in clinical trials, most applications still use traditional culture media containing fetal bovine serum or serum-free media that contain serum albumin, insulin, and transferrin. The ill-defined and variable nature of traditional culture media remains a challenge and has created a need for better defined xeno-free culture media to meet the regulatory and long-term safety requirements for cell-based therapies. We developed and tested a serum-free and xeno-free culture medium (SFM-XF) using human bone marrow- and adipose-derived MSCs by investigating primary cell isolation, multiple passage expansion, mesoderm differentiation, cellular phenotype, and gene expression analysis, which are critical for complying with translation to cell therapy. Human MSCs expanded in SFM-XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of MSCs expanded in traditional serum-containing culture medium (SCM). To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded in SFM-XF and SCM were compared, revealing relatively similar expression profiles. In addition, the SFM-XF supported the isolation and propagation of human MSCs from primary human marrow aspirates, ensuring that these methods and reagents are compatible for translation to therapy. The SFM-XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to serum-containing media for the production of large scale, functionally competent MSCs for future clinical applications.

Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions.

The immunomodulatory properties of mesenchymal stem cells (MSCs) make them attractive therapeutic agents for a wide range of diseases. However, the highly demanding cell doses used in MSC clinical trials (up to millions of cells/kg patient) currently require labor intensive methods and incur high reagent costs. Moreover, the use of xenogenic (xeno) serum-containing media represents a risk of contamination and raises safety concerns. Bioreactor systems in combination with novel xeno-free medium formulations represent a viable alternative to reproducibly achieve a safe and reliable MSC doses relevant for cell therapy. The main goal of the present study was to develop a complete xeno-free microcarrier-based culture system for the efficient expansion of human MSC from two different sources, human bone marrow (BM), and adipose tissue. After 14 days of culture in spinner flasks, BM MSC reached a maximum cell density of (2.0±0.2)×105 cells·mL⁻¹ (18±1-fold increase), whereas adipose tissue-derived stem cells expanded to (1.4±0.5)×105 cells·mL⁻¹ (14±7-fold increase). After the expansion, MSC expressed the characteristic markers CD73, CD90, and CD105, whereas negative for CD80 and human leukocyte antigen (HLA)-DR. Expanded cells maintained the ability to differentiate robustly into osteoblast, adipocyte, and chondroblast lineages upon directed differentiation. These results demonstrated the feasibility of expanding human MSC in a scalable microcarrier-based stirred culture system under xeno-free conditions and represent an important step forward for the implementation of a Good Manufacturing Practices-compliant large-scale production system of MSC for cellular therapy.

Mesenchymal stem cell assays and applications.

Research on mesenchymal stem cells (MSC) is progressing with increasing popularity. Currently there are a significant number of clinical trials exploring the use of MSCs for the treatment of various diseases including graft-versus-host disease, Crohn's disease, myocardial infarction, stroke, bone defects, diabetes, and wound repair (www.-clinicaltrials.gov). At the same time, there are questions associated with MSCs in terms of their isolation, culture expansion, phenotype, multipotential differentiation, and transplantation efficiency. This chapter outlines the current status of the field and emphasizes the need for clearly defined protocols to better define the function and use of MSCs in cell therapy.

A novel serum-free medium for the expansion of human mesenchymal stem cells.

INTRODUCTION: Human multipotent mesenchymal stem cell (MSC) therapies are being tested clinically for a variety of disorders, including Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, and cartilage defects. However, despite the remarkable clinical advancements in this field, most applications still use traditional culture media containing fetal bovine serum. The ill-defined and highly variable nature of traditional culture media remains a challenge, hampering both the basic and clinical human MSC research fields. To date, no reliable serum-free medium for human MSCs has been available. METHODS: In this study, we developed and tested a serum-free growth medium on human bone marrow-derived MSCs through the investigation of multiple parameters including primary cell isolation, multipassage expansion, mesoderm differentiation, cellular phenotype, and gene-expression analysis. RESULTS: Similar to that achieved with traditional culture medium, human MSCs expanded in serum-free medium supplemented with recombinant human platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF), and transforming growth factor (TGF)-beta1 showed extensive propagation with retained phenotypic, differentiation, and colony-forming unit potential. To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded under serum-free and serum-containing conditions were compared, revealing similar expression profiles. In addition, the described serum-free culture medium supported the isolation of human MSCs from primary human marrow aspirate with continual propagation. CONCLUSIONS: Although the described serum-free MSC culture medium is not free of xenogeneic components, this medium provides a substitute for serum-containing medium for research applications, setting the stage for future clinical applications.

Serum-free, xeno-free culture media maintain the proliferation rate and multipotentiality of adipose stem cells in vitro.

BACKGROUND AIMS: Human adipose stem cells (ASC) are an abundant, readily available population of multipotent progenitor cells that reside in adipose tissue. ASC have been shown to have therapeutic applicability in pre-clinical studies, but a standardized expansion method for clinical cell therapy has yet to be established. Isolated ASC are typically expanded in medium containing fetal bovine serum (FBS); however, sera and other culturing reagents of animal origin in clinical therapy pose numerous safety issues, including possible infections and severe immune reactions. METHODS: To identify optimal conditions for ex vivo expansion of ASC, the effects of seven serum-free (SF) and xeno-free (XF) media were investigated with both FBS and allogeneic human serum (alloHS; as a control media). Surface marker expression, proliferation, morphology and differentiation analyzes were utilized for investigating the effects of media on ASC. RESULTS: The proliferation and morphology analysis demonstrated significant differences between ASC cultured in SF/XF culture media compared with serum-containing culture media, with medium prototype StemPro MSC SFM XenoFree providing significantly higher proliferation rates than ASC cultured in media containing serum, while still maintaining the differentiation potential and surface marker expression profile characteristic of ASC. CONCLUSIONS: Looking forward, fully defined XF media formulations will provide the means for the development and approval of safer clinical cell therapy treatments. However, to fully recognize the capacity of these XF culture media, further pre-clinical safety and efficacy studies must be performed.

A simplified culture and polymerase chain reaction identification assay for quality control performance testing of stem cell media products.

BACKGROUND AIMS: With the growing use of stem cell media technologies in research and clinical settings, there has been an increased demand for validated cell-based quality control tools that can first, routinely test performance of stem cell media products, second, verify stem cell line identity, and third, demonstrate differentiation potential. As a significant amount of time and effort is required to verify these aspects separately, especially with classic functional stains that take as along as 28 days to perform, there is a need for a quick, sensitive and validated assay with short turn around time. METHODS: Culture, gene microarray and polymerase chain reaction (PCR) methodologies were utilized in the design, development and testing of a standardized performance assay for the expansion, identity and differentiation potential of human multipotent mesenchymal stromal cells (MSC). RESULTS: A simplified culture- and PCR-based assay was validated and transferred into a quality control setting for performance testing of human MSC under uninduced and adipogenesis-induced conditions. CONCLUSIONS: An effective strategy has been demonstrated for identifying candidate genes, validating a gene of interest and creating an inexpensive low-technology PCR assay for distinguishing uninduced and early stage differentiating stem cells. This approach extends published criteria guidelines for routinely detecting uninduced human MSC and their differentiated progeny.

Neural transplantation and stem cells.

Recent results have raised important questions on our ability to amplify stem cell populations in sufficient numbers as to be useful for therapy. Several reports have indicated that human stem cell populations harvested from the adult have low or undetectable telomerase levels, age in culture, and may not be propagated indefinitely. Other groups have shown that stem cells age and as such, their properties will have changed depending on the age of the individual from which they are harvested, and the time for which they are propagated in culture. Other groups have shown that cells maintained in culture may undergo alterations as they are propagated, and that these alterations may alter the predicted behavior of stem cells. Yet others have shown that human cells differ from their counterparts in other species in significant ways and have identified important difficulties in assessing cells in a xeno environment. Clinical colleagues have identified issues of variability and difficulties in the long-term follow-up that is being requested. Researchers in the stem cell field focused on translational work need to develop a practical plan that takes into account such difficulties while developing manufacturing protocols, designing animal studies, or developing trial protocols. Such proactive planning will be critical in ensuring a successful transition from the bench to the clinic.

Human cord blood stem cell applications in cell therapy.

Human umbilical cord blood (UCB) is a valuable alternative source of ethically acceptable, clinically competent stem cells that is most likely closest to embryonic stem cells. Development of reliable methods for the expansion of cord blood stem cells is critical to ensure their clinical application. In the present article, advances in cord blood stem cell isolation, culture expansion methods through co-culture with human mesenchymal stem cells, culture optimization techniques with defined media and cord blood stem cell banking aspects have been reviewed. Refined methods of isolation as well as defined culture conditions of expansion that favor retention of stem cell phenotype and proper cryogenic storage can significantly increase the use of cord blood stem cells in human cell therapy applications.