Lindvall, O; Kokaia, Z; Martinez-Serrano, A

Nature Medicine [Nat. Med.]. Vol. 10, suppl., pp. S42-S50. Jul 2004.

Recent progress shows that neurons suitable for transplantation can be generated from stem cells in culture, and that the adult brain produces new neurons from its own stem cells in response to injury. These findings raise hope for the development of stem cell therapies in human neurodegenerative disorders. Before clinical trials are initiated, we need to know much more about how to control stem cell proliferation and differentiation into specific phenotypes, induce their integration into existing neural and synaptic circuits, and optimize functional recovery in animal models closely resembling the human disease.

Silani, V; Cova, L; Corbo, M; Ciammola, A; Polli, E

Lancet [Lancet]. Vol. 364, no. 9429, pp. 200-202. 0-16 Jul 2004.

With the lack of effective drug treatments for amyotrophic lateral sclerosis (ALS), and compelling preclinical data, stem-cell research has highlighted this disease as a candidate for stem-cell treatment. Stem-cell transplantation is an attractive strategy for neurological diseases and early successes in animal models of neurodegnerative disease generated optimism about restoring function or delaying degeneration in human beings. The restricted potential of adult stem cells has been challenged over the past 5 years by reports on their ability to acquire new unexpected fates beyond their embryonic lineage (transdifferentiation). Therefore, autologous or allogeneic stem cells, undifferentiated or transdifferentiated and manipulated epigenetically or genetically, could be a candidate source for local or systemic cell-therapies in ALS. Albert Clement and colleagues showed that in SOD1G93A chimeric mice, motorneuron degeneration requires damage from mutant SOD1 acting in non-neuronal cells. Wild-type non-neuronal (glial) cells could delay degeneration and extend survival of mutant-expressing motorneurons. Letizia Mazzini and colleagues injected autologous bone-marrow-derived stem cells into the spinal cord of seven ALS patients. These investigators reported that the procedure had a reasonable margin of clinical safety. The success of cell-replacement therapy in ALS will depend a lot on preclinical evidence, because of the complexity and precision of the pattern of connectivity that needs to be restored in degenerating motoneurons. Stem-cell therapy will need to be used with other drugs or treatments, such as antioxidants and/or infusion of trophic molecules.

Gerecht-Nir, Sharon; Cohen, Smadar; Itskovitz-Eldor, Joseph

Biotechnology and Bioengineering [Biotechnol. Bioeng.]. Vol. 86, no. 5, pp. 493-502. 2004.

The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy and tissue engineering depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe for the first time the formation of differentiating human embryoid bodies (hEBs) in rotating bioreactors to try and control their agglomeration. The efficacy of the dynamic process compared to static cultivation in Petri dishes was analyzed with respect to the yield of hEB formation and differentiation. Quantitative analyses of hEBs, DNA and protein contents, and viable cell concentration, as measures for culture cellularity and scale-up, revealed 3-fold enhancement in generation of hEBs compared to the static culture. Other metabolic indices such as glucose consumption, lactic acid production, and pH pointed to efficient cell expansion and differentiation in the dynamic cultures. The type of rotating vessel had a significant impact on the process of hEB formation and agglomeration. In the slow turning lateral vessel (STLV), hEBs were smaller in size and no large necrotic centers were seen, even after 1-month cultivation. In the high aspect rotating vessel (HARV), hEB agglomeration was massive. The appearance of representative tissues derived from the three germ layers as well as primitive neuronal tube organization, blood vessel formation, and specific-endocrine secretion indicated that the initial developmental events are not altered in the dynamically formed hEBs. Collectively, our study defines the culture conditions in which control over the aggregation of differentiating hESCs is obtained, thus enabling scaleable cell production for clinical and industrial applications.

Kosaka, Y; Kobayashi, N*; Fukazawa, T; Totsugawa, T; Maruyama, M; Yong, C; Arata, T; Ikeda, H; Kobayashi, K*; Ueda, T; Kurabayashi, Y; Tanaka, N

Artificial Organs [Artif. Organs]. Vol. 28, no. 3, pp. 271-277. Mar 2004.

Embryonic stem (ES) cells are widely used in therapeutic research as an unlimited source of cell therapy. Therefore, it is of great value to find a way to efficiently manipulate ES cells. HIV-1-derived lentiviral vectors are now considered to be an efficient vehicle for delivering genes into a variety of cells. In this study, we examined the efficacy of lentivirus-based gene delivery into mouse ES (mES) cells. Recombinant HIV-I-based lentiviral vectors Lt-GFP, expressing green fluorescent protein (GFP), and Lt-LacZ, expressing E. coli LacZ gene in conjunction with neomycin resistance gene, were generated using a FuGENE 6 transduction method and used for transducing ES cells derived from 129Sv mice. Lentiviral transduction efficacy was evaluated by GFP expression assay using flow cytometry and by X-gal staining. The in vivo potential of developing teratoma of such transduced mES cells was examined in severe combined immunodeficiency (SCID) mice. FuGENE 6 showed no considerable transduction-associated cytotoxicity. The expression rate of GFP and LacZ of mES cells increased on a multiplicity of infection (MOI)-dependent manner with the amount of Lt-GFP and Lt-LacZ used. Approximately 42% of mES cells were positive for GFP after infection of Lt-GFP at an MOI of 30. Notably, after G418 selection, nearly 100% of Lt-LacZ-transduced mES cells were positive for LacZ and formed teratomas in SCID mice. This work demonstrates that HIV-I-based lentiviral vectors are capable of transducing mES cells. Lentiviral vectors may facilitate an advance in the field of gene transfer and expression in various types of ES cells, including human ES cells.

Salgar, SK; Yang, D; Ruiz, P; Miller, J; Tzakis, AG

Human Gene Therapy [Hum. Gene Ther.]. Vol. 15, no. 2, pp. 131-144. Feb 2004.

The Epstein-Barr virus-encoded protein BCRF1 (viral interleukin [vIL]-10) is a biologically active homologue of cellular interleukin (IL)-10. In this study, a novel gene therapy approach to prolong allograft survival was designed. Autologous (syngeneic) hematopoietic progenitor/stem cell-enriched (HSC; lineage super(-ve)) population derived from CBA/J mouse bone marrow were transduced with retrovirus encoding vIL-10 gene (vIL-10-HSC), ex vivo; vIL-10-HSC were injected (4-6 x 10 super(6) cells intravenously) into lethally (9.5 Gy) or sublethally (4 Gy) irradiated CBA/J mice. Six weeks after vIL-10-HSC administration, vascular heterotopic heart (C57BL/6) transplantation was performed. Ex vivo, the vIL-10-HSC produced 5.4 plus or minus 0.5 ng of vIL-10 protein/2 x 10 super(5) cells per 24 hr. In vivo, serum vIL-10 production was 187 plus or minus 205 pg/ml during 3-10 weeks after vIL-10-HSC administration. Cardiac allograft survival was prolonged (p < 0.004) in lethally (71 plus or minus 40 days) and sublethally (114 plus or minus 15 days) irradiated mice that received vIL-10-HSC compared to controls that received unengineered (UE) HSC or vector DNA-engineered HSC (12-16 days). However, secondary skin graft (C57BL/6) survival was not prolonged in cardiac allograft-tolerant animals. In the vIL-10-HSC-administered group, graft histopathology demonstrated mild arteritis/venulitis (grade 0.7) and rejection (grade 1.0). Intra-graft expression of costimulatory molecules (B7.1, B7.2), cytokines (IL-2, IL-4, mIL-10, interferon [IFN]- gamma ), and inducible nitric oxide synthase (iNOS) molecules was markedly lower in vIL-10-HSC-treated tolerant grafts that survived more than 100 days compared to vector DNA-HSC- or UE-HSC-treated controls. Furthermore, T lymphocytes derived from vIL-10-HSC-treated tolerant recipients demonstrated hyporeactivity to donor antigens in mixed lymphocyte cultures. Administration of autologous vIL-10-engineered HSC prior to organ transplantation prolonged cardiac allograft survival significantly.

Murphy, JM; Fink, DJ; Hunziker, EB; Barry, FP*

Arthritis & Rheumatism [Arthritis Rheum.]. Vol. 48, no. 12, pp. 3464-3474. Dec 2003.

To explore the role that implanted mesenchymal stem cells may play in tissue repair or regeneration of the injured joint, by delivery of an autologous preparation of stem cells to caprine knee joints following induction of osteoarthritis (OA). Adult stem cells were isolated from caprine bone marrow, expanded in culture, and transduced to express green fluorescent protein. OA was induced unilaterally in the knee joint of donor animals by complete excision of the medial meniscus and resection of the anterior cruciate ligament. After 6 weeks, a single dose of 10 million autologous cells suspended in a dilute solution of sodium hyaluronan was delivered to the injured knee by direct intraarticular injection. Control animals received sodium hyaluronan alone. In cell-treated joints, there was evidence of marked regeneration of the medial meniscus, and implanted cells were detected in the newly formed tissue. Degeneration of the articular cartilage, osteophytic remodeling, and subchondral sclerosis were reduced in cell-treated joints compared with joints treated with vehicle alone without cells. There was no evidence of repair of the ligament in any of the joints. Local delivery of adult mesenchymal stem cells to injured joints stimulates regeneration of meniscal tissue and retards the progressive destruction normally seen in this model of OA.

Hassink, RJ; Dowell, JD; De la Riviere, AB; Doevendans, PA; Field, LJ

Trends in Molecular Medicine [Trends Mol. Med.]. Vol. 9, no. 10, pp. 436-441. Oct 2003.

Recent experimental and clinical observations have suggested that cell transplantation could be of therapeutic value for the treatment of heart disease. This approach was based on the idea that transplanted donor cardiomyocytes would integrate with the host myocardium and thereby directly contribute to cardiac function. Surprisingly, the observation that non- cardiomyogenic cells could also improve cardiac function indicates that functional integration of donor cells might not be required to achieve a beneficial effect. More recently, several observations have suggested the presence of a greater than anticipated developmental repertoire in adult-derived stem cells, which, if further validated, would offer unprecedented opportunities for the restoration of cardiac function in diseased hearts. Here, we discuss current issues regarding the potential use of stem cell transplantation for the treatment of ischemic heart disease.

Vrana, KE; Hipp, JD; Goss, AM; McCool, BA; Riddle, DR; Walker, SJ; Wettstein, PJ; Studer, LP; Tabar, V; Cunniff, K; Chapman, K; Vilner, L; West, MD; Grant, KA; Cibelli, JB

Proceedings of the National Academy of Sciences, USA [Proc. Natl. Acad. Sci. USA]. Vol. 100, suppl. 1, pp. 11911-11916. 30 Sep 2003.

Parthenogenesis is the biological phenomenon by which embryonic development is initiated without male contribution. Whereas parthenogenesis is a common mode of reproduction in lower organisms, the mammalian parthenote fails to produce a successful pregnancy. We herein describe in vitro parthenogenetic development of monkey (Macaca fascicularis) eggs to the blastocyst stage, and their use to create a pluripotent line of stem cells. These monkey stem cells (Cyno-1 cells) are positive for telomerase activity and are immunoreactive for alkaline phosphatase, octamer-binding transcription factor 4 (Oct-4), stage- specific embryonic antigen 4 (SSEA-4), tumor rejection antigen 1-60 (TRA 1-60), and tumor rejection antigen 1-81 (TRA 1-81) (traditional markers of human embryonic stem cells). They have a normal chromosome karyotype (40 + 2) and can be maintained in vitro in an undifferentiated state for extended periods of time. Cyno-1 cells can be differentiated in vitro into dopaminergic and serotonergic neurons, contractile cardiomyocyte-like cells, smooth muscle, ciliated epithelia, and adipocytes. When Cyno-1 cells were injected into severe combined immunodeficient mice, teratomas with derivatives from all three embryonic germ layers were obtained. When grown on fibronectin/laminin-coated plates and in neural progenitor medium, Cyno-1 cells assume a neural precursor phenotype (immunoreactive for nestin). However, these cells remain proliferative and express no functional ion channels. When transferred to differentiation conditions, the nestin-positive precursors assume neuronal and epithelial morphologies. Over time, these cells acquire electrophysiological characteristics of functional neurons (appearance of tetrodotoxin-sensitive, voltage-dependent sodium channels). These results suggest that stem cells derived from the parthenogenetically activated nonhuman primate egg provide a potential source for autologous cell therapy in the female and bypass the need for creating a competent embryo.

Perrier, AL; Studer, L*

Seminars in Cell & Developmental Biology [Semin. Cell Dev. Biol.]. Vol. 14, no. 3, pp. 181-189. Jun 2003.

Midbrain dopamine (DA) neurons play an essential role in modulating motor control, and their degeneration is the hallmark feature of Parkinson's disease (PD). In vitro production of DA neurons provides insight into the mechanisms that control cell fate choice, and offers an alternative to the use of fetal tissue for experimental cell replacement in PD. Here we will review the advantages and disadvantages of the various renewable cell sources and protocols tested, and discuss their relevance for basic studies and for cell therapy.