Mohan C. Vemuri

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.

Generation of Human-Induced Pluripotent Stem Cells by a Nonintegrating RNA Sendai Virus Vector in Feeder-Free or Xeno-Free Conditions

The generation of induced pluripotent stem cells (iPSCs) from somatic cells has enabled the possibility of providing unprecedented access to patient-specific iPSC cells for drug screening, disease modeling, and cell therapy applications. However, a major obstacle to the use of iPSC for therapeutic applications is the potential of genomic modifications caused by insertion of viral transgenes in the cellular genome. A second concern is that reprogramming often requires the use of animal feeder layers and reagents that contain animal origin products, which hinder the generation of clinical-grade iPSCs. Here, we report the generation of iPSCs by an RNA Sendai virus vector that does not integrate into the cells genome, providing transgene-free iPSC line. In addition, reprogramming can be performed in feeder-free condition with StemPro hESC SFM medium and in xeno-free (XF) conditions. Generation of an integrant-free iPSCs generated in xeno-free media should facilitate the safe downstream applications of iPSC-based cell therapies.

Critical steps in the isolation and expansion of adipose-derived stem cells for translational therapy

Since the discovery of adipose-derived stem cells (ASCs), there have been high expectations of their putative clinical use. Recent advances support these expectations, and it is expected that the transition from pre-clinical and clinical studies to implementation as a standard treatment modality is imminent. However ASCs must be isolated and expanded according to good manufacturing practice guidelines and a basic assurance of quality, safety, and medical effectiveness is needed for authorisation by regulatory agencies, such as European Medicines Agency and US Food and Drug Administration. In this review, a collection of studies investigating the influence of different steps of the isolation and expansion protocol on the yield and functionality of ASCs has been presented in an attempt to come up with best recommendations that ensure potential beneficial clinical outcome of using ASCs in any therapeutic setting. If the findings confirm the initial observations of beneficial effects of ASCs, the path is paved for implementing these ASC-based therapies as standard treatment options.

Good cell culture practice for stem cells and stem-cell-derived models

The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.

A xeno-free culturing protocol for pluripotent stem cell-derived oligodendrocyte precursor cell production

AIM To show that human embryonic stem cells (hESCs) can be efficiently differentiated into oligodendrocyte precursor cells (OPCs) in a xeno-free medium with a specific medium supplement and specific human recombinant growth factors. MATERIALS & METHODS The xeno-free OPC-differentiation medium for pluripotent stem cells was developed by using StemPro® neural stem cell xeno-free medium supplement together with human recombinant growth factors SHH, PDGF-AA, IGF-1, EGF, basic FGF and CNTF, in addition to RA, T3, human laminin and ascorbic acid. We analyzed the differentiated hESC-derived OPCs and oligodendrocytes with quantitative real-time (RT)-PCR, RT-PCR, flow cytometry and immunocytochemistry, and we performed NG2-positive selection for OPC cultures with fluorescence-activated cell sorting. RESULTS Based on quantitative RT-PCR analysis, OPCs after 9 weeks of differentiation in xeno-free medium expressed OLIG2, SOX10 and NKX2.2 at elevated levels compared with control conditions. According to the flow cytometric analysis, the cells expressed A2B5 (>70%) and NG2 (40-60%) at 5 weeks time point whereas maturing oligodendrocytes expressed O4 (60-80%) at 11 weeks time point. In addition, hESC-derived OPC populations were purified based on NG2-positive selection using fluorescence-activated cell sorting. NG2-positive OPC populations survived and differentiated further into O4 expressing oligodendrocytes in xeno-free medium, and the sorted cell populations were free from pluripotent Tra1-81 and Oct-4 -positive cells. CONCLUSIONS This study confirms that the xeno-free culturing method can support the differentiation and purification of hESC-derived OPC populations and provides an initial step toward safe cell graft production for the future clinical applications.

Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells

The potential applications of human embryonic stem cells (hESCs) in regenerative medicine and developmental research have made stem cell biology one of the most fascinating and rapidly expanding fields of biomedicine. The first clinical trial of hESCs in humans has begun, and the field of stem cell therapy has just entered a new era. Here, we report seven hESC lines (SEES-1, -2, -3, -4, -5, -6, and -7). Four of them were derived and maintained on irradiated human mesenchymal stem cells (hMSCs) grown in xenogeneic-free defined media and substrate. Xenogeneic-free hMSCs isolated from the subcutaneous tissue of extra fingers from individuals with polydactyly showed appropriate potentials as feeder layers in the pluripotency and growth of hESCs. In this report, we describe a comprehensive characterization of these newly derived SEES cell lines. In addition, we developed a scalable culture system for hESCs having high biological safety by using gamma-irradiated serum replacement and pharmaceutical-grade recombinant basic fibroblast growth factor (bFGF, also known as trafermin). This is first report describing the maintenance of hESC pluripotency using pharmaceutical-grade human recombinant bFGF (trafermin) and gamma-irradiated serum replacement. Our defined medium system provides a path to scalability in Good Manufacturing Practice (GMP) settings for the generation of clinically relevant cell types from pluripotent cells for therapeutic applications.

Stem cell assays

Book Title: Stem Cell Assays Publisher: Humana Press Editor : Mohan C. Vemuri Series Editor : John Walker Series : Methods in Molecular Biology Date of Release: Late 2006 / Early 2007 Contents Preface Contributors Guide to the companion CD 1 Derivation of Human Embryonic Stem Cells in xeno-free conditions Mohan C. Vemuri, Tim Schimmel, Pere Colls, Santiago Munne and Jacques Cohen 2 Feeder Layer Free Culture System For Human Embryonic Stem Cells Michal Amit 3 Digital Imaging of Stem Cells by Electron Microscopy Henry Sathananthan and Stefania Nottola 4 A Controlled Cooling Protocol For Cryopreservation Of Human And Non-Human Primate Embryonic Stem Cells Carol B. Ware and Szczepan Baran 5 Cell Surface Markers In Human Embryonic Stem Cells Raj R. Rao, Alison Venable Johnson and Steven L. Stice 6 Generation Of Monoclonal Antibody Library Against Human Embryonic Stem Cells Micha Drukker, Christina Muscat, Irving L. Weissman 7 Analytical Methods for Cancer Stem Cells - Review chapter Vinagolu K. Rajasekhar 8 Micro RNA Profiling: An Easy and Rapid Method to Screen and Characterize Stem Cell Populations Uma Lakshmipathy, Bradley Love, Christopher Adams, Bhaskar Thyagarajan, and Jonathan D. Chesnut 9 Gene Transfer Via Nucleofection into Adult And Embryonic Stem Cells Uma Lakshmipathy, Shannon Buckley and Catherine Verfaillie 10 RNAi-Knockdown Of Transcription Factor Pu.1 In The Differentiation Of Mouse Embryonic Stem Cells Gang-Ming Zou, Meredith A. Thompson and Mervin C. Yoder 11 StemBase - A Resource for the Analysis of Stem Cell Gene Expression Data - Review chapter Christopher J. Porter, Gareth A. Palidwor, Reatha Sandie, Paul M. Krzyzanowski, Enrique M. Muro, Carolina Perez-Iratxeta, Miguel A. Andrade-Navarro 12 Isolation Of Stem Cells From Human Umbilical Cord Blood Nishant P. Reddy, Mohan C. Vemuri and Reddanna Pallu 13 Ex Vivo Expansion Of Hematopoietic Stem Cells From Human Cord Blood In Serum-Free Conditions Chao-Ling Yao and Shiaw-Min Hwang 14 Hematopoietic Colony Forming Cell Assays Carla Pereira, Emer Clarke and Jackie Damen 15 Assays For Alloreactive Responses By PCR Patrick Stordeur 16 Immune Properties of Mesenchymal Stem Cells Review chapter Panagiota A. Sotiropoulou and Michael Papamichail 17 Clinical Grade Expansion of Human Bone Marrow Mesenchymal Stem Cells Panagiota A. Sotiropoulou, Sonia A. Perez and Michael Papamichail 18 Adenoviral Transduction of Mesenchymal Stem Cells Pablo Bosch and Steven L. Stice 19 Directed Differentiation of Human Embryonic Stem Cells to Dendritic Cells Maxim A. Vodyanik and Igor I. Slukvin 20 Insulin Producing Cells From Embryonic Stem Cells: Experimental Considerations Enrique Roche, Roberto Ensenat-Waser, Nestor Vicente-Sala, Alfredo Santana, Martin Zenke and Juan Antonio Rei 21 Efficient Generation of Dopamine Neurons from Human Embryonic Stem (hES) Cells Chang-Hwan Park and Sang-Hun Lee 22 Isolation Of Oligodendroglial Cells From Cultured Neural Stem/Progenitors Frank Zeigler and Stephen G. Hall 23 Differentiation of Human Embryonic Stem Cells towards the Chondrogenic Lineage Wei Seong Toh, Zheng Yang Boon Chin Heng and Tong Cao 24 Cartilage Tissue Engineering: Directed differentiation of embryonic stem cells in Three-Dimensional Hydrogel Culture Nathaniel S. Hwang, Shyni Varghese and Jennifer Elisseeff

Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.

Hypoxia enhances the wound-healing potential of adipose-derived stem cells in a novel human primary keratinocyte-based scratch assay

Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) promote the healing of chronic wounds, and that the exposure of ASCs to hypoxia enhances their wound healing effect. To aid the translation of these findings into clinical use, robust wound models are necessary to explore each aspect of wound healing. The aspect of re-epithelization is often studied in a scratch assay based on transformed keratinocytes. However, there are concerns regarding the validity of this model, since these cell lines differ from normal keratinocytes, both in terms of proliferative capacity and differentiation, and sensitivity to environmental cues. In this study, the main challenge of using primary keratinocytes to examine the effects of ASCs was identified to be their different requirements for calcium in the culture media. We confirmed that a high calcium content led to morphological and cytoskeletal changes in primary keratinocytes, and demonstrated that a low calcium content compromised the growth of ASCs. We found that it is possible to perform the wound healing assay with primary keratinocytes, if the conditioned media from the ASCs is dialyzed to reduce the calcium concentration. Additionally, using this model of re-epithelization, conditioned media from normoxic ASCs was shown to markedly increase the rate of wound closure by primary keratinocytes, and this effect was significantly enhanced with media from the hypoxia-exposed ASCs. These findings, which are in line with the observations from previous in vivo studies, highlight the validity of this modified assay to investigate the wound healing properties of ASCs in vitro.

Pluripotent Stem Cells

Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass (ICM) of the developing embryo. hESCs culture as cell lines in vitro and possess great potential in such research fi elds as developmental biology and cell-based therapy, as well as such industrial purposes as drug screening and toxicology. When ESCs were fi rst derived by Thomson and colleagues, traditional methods of immunostaining and culturing, using primary mouse embryonic fi broblasts and medium supplemented by serum were used. Considerable efforts have since led to improved methods for isolating new lines in de fi ned and reproducible conditions. This chapter discusses sources for embryos for ESC isolation, commonly used methods for deriving hESC lines, and a number of possible culture systems.