1 STATE OF THE ART 3B-PL2 ISBT Science Series (2011) 6, ª 2011 The Author(s). ISBT Science Series ª 2011 International Society of Blood Transfusion Human cord blood reprogrammed into embryonic-like stem cells A. Giorgetti, R. Fazzina, M. Li & J. C. I. Belmonte Salk Institute for Biological Studies, La Jolla, USA Embryonic stem (ES) cell therapies are often promoted as the optimal stem cell source for regenerative medicine applications. Although the first clinical trail involving hes progenitors has been approved, ES cell applications are currently limited by ethical, political and regulatory hurdles. In addition, the use of hes cell-derived progenitors has been fraught with difficulties associated with immunological incompatibility, due in part to the increase in expression of major histocompatibility complex molecules during differentiation of hes cells. Induced pluripotent stem (ips) cells could solve both the ethical problem of human embryo use and the immunological rejection problem. Patient-specific ipsc have been hailed as an enormous development for regenerative medicine since transplantation of the differentiated progeny of these individual ipsc should not be subject to immune rejection when transplanted back into a patient. However, in many instances, a ready to use approach could be desirable, such as for cell therapy of acute conditions or when the patient s somatic cells are altered as a consequence of a chronic disease or ageing. An alternative could be the generation of healthy ipsc lines with a wide genetic variety rather than specific-patient ipsc, which will enable broader immune histocompatibility. However, this is not presently feasible as many thousand of hips-cell lines would be required to ensure sufficient diversity. Cord blood (CB) stem cells could represent a new source of cells for the generation of clinically sound ipsc in an allogenic setting. For example, a bank of CB-iPSC derived from selected donors homozygous for common human leukocyte antigen (HLA) haplotypes, could significantly reduce the number of CB-iPSC lines needed to provide a perfect HLA match for a large percentage of the population. We have recently reported that the overexpression of only two transcription factors, OCT4 and SOX2, are sufficient to reprogram CB CD133+ cells faster than fibroblasts and keratinocytes. CB-iPSC showed a differential potential similar to human embryonic stem cells in vitro and in vivo. Following specific in vitro differentiation protocols, CBiPSC gave rise also to specialized cell types such as rhythmically beating cardiomyocytes and dopaminergic neurons. The generation of CB-iPSC lines from thawed CB units, that had been stored frozen for more than 8 years, has excluded the possibility that the standard cryopreservation protocol could affect the reprogramming process. In addition, we have demonstrated that CB cells were properly reprogrammed into pluripotent stem cells from both the expression and the epigenetic point of view. However, a number of technical issues need to be resolved before the ips technology can be used in a clinical setting. These include the establishment of efficient reprogramming strategies that do not result in genetically modified cells as well as the development of robust protocols for differentiating ipsc to self-renewing stem cells and lineage-committed cells. Ultimately, these methods must be adapted to the generation of ipsc under good manufacturing practice conditions. Correspondence: J.C. Izpisua Belmonte, Salk Institute for Biological Studies, La Jolla, USA 107
2 108 A. Giorgetti et al. Introduction A primary goal of regenerative medicine is to produce new cells to repair or replace diseased and damaged tissues. The discovery of human embryonic stem cells (hesc) opened up the possibility for the application of human pluripotent stem cells in transplantation therapy, drug screening, and toxicology studies, as well as functional genomics and proteomics research [1,2]. Recently, the Food and Drug Administration (FDA) has approved clinical trials involving hes cell progenitors for the treatment of patients with spinal cord injury and congenital retinopathies. However, the use of hes cell-derived progenitors has been fraught difficulties associated with the fact that they could form tumours and they are ethically controversial because they originate from human embryos. In addition, problems related to the immune rejection represent another limit for the application of hesc in transplantation. It would be difficult to obtain the large numbers of embryos required to cover a wide range of human leukocyte antigen (HLA) haplotype for close tissue matching to patients . Reprogramming somatic cells into pluripotent cells could address these limitations. Among the many approaches proposed, such as somatic cell nuclear transfer (SCNI) [4,5] or cell-cell fusion of somatic cells and hescs [6,7], the generation of induced pluripotent stem cells (ips- Cs) represents the most promising reprogramming technique for human cells [8,9]. So far, human ipscs have been generated from numerous somatic cell types  and recently our group have described the possibility to generate ipscs also from Cord BLOOD (CB) stem cells . Generation of human ips cells A huge amount of effort has gone into developing functional equivalents of hesc that do not involve the destruction of human embryos or eggs. The most straight forward way for the induction of pluripotent stem cells was pioneered by Takahashi and Yamanaka . They demonstrated that retroviral-mediated overexpression of just four transcription factors (OCT4, SOX2, KLF4 and c-myc), was sufficient to reprogramme murine fibroblasts to an embryonic-like state. Lately the same group, contemporaneously with other groups [12,13], succeeded in generating ipscs starting from human somatic cells. The ipscs are similar to nesc in morphology, proliferation, differentiation and teratoma formation. However, small differences in gene expression and DNA methylation patterns between ipscs and nesc have been observed . In general, the efficiency of ipscs generation is low and the KINETIC of reprogramming varies with different target cell populations. Although human ipscs have been generated using different type of somatic cells, such as skin fibroblasts and keratinocytes, neural stem cells is hepatocytes and blood cells, it is still under debate what the best source to derive ipscs [9,15,16]. The possibility of reprogramming mature somatic cells has allowed for the production of pluripotent cells that carry the specific GENOME of individuals, providing an unprecedented experimental platform to model human diseases. Patient-specific ipscs could help to establish in vitro disease models and might lead to the discovery of drugs for treating patients. This is particularly important for diseases that lack adequate in vitro or animal models. Therefore, patient-specific ipscs have been hailed as an enormous development for regenerative medicine as transplantation of the differentiated progeny of these individual ipscs should not be subject to immune rejection when transplanted back into a patient. So far, disease-specific ipsc lines have already generated from fibroblasts of patients with amyotrophic later sclerosis , juvenile onset type-1 diabetes mellitus , Parkinson s disease and spinal muscular atrophy , as well as b-thalassemia and Fanconi anaemia . However, in many instances, a ready to use approach could be desirable, such as for cell therapy of acute conditions or when the patient s somatic cells are altered as a consequence of a chronic disease or ageing. An option could be the generation of healthy ipsc lines with a wide genetic variety rather than specific-patient ipscs, which will enable broader immune histocompatibility. CB stem cells, currently widely used as a source of haematopoietic stem cells for transplantation, appear ideally suited for the generation of clinically sound ipscs in an allogenic setting. Cord blood ipscs A general impression is that the differentiation status of the target cells influences the reprogramming process, as illustrated by the fact that haematopoietic stem cells are more efficiently reprogrammed than terminally differentiated B and T-lymphocytes . CB cells have several distinct advantages over other haematopoietic stem cell sources: they can be cryopreserved and stored for years without loss of viability and selfrenewal capability. In comparison with stem cell population derived from Bone Marrow (BM) or mobilized peripheral blood (mpb), they have greater tolerance across 1 or 2 HLA mismatching and reduced risk of viral contamination and graft-versus-host disease during allogenic transplantation. Moreover, CB cells can be harvested without any risk for the donor and there are easily characterized and banked. Public banking of CB units has been established throughout the world, providing easy access to CB cells from worldwide registries . Currently the clinical use of ipscs is limited, because they are generated from somatic cells that have accumulated genetic mutations
3 Human cord blood reprogrammed 109 (a) (b) (c) (d) (e) Fig. 1 Images related to ips cells characterization derived using a fresh cord blood unit: (a) Images of established ips cells before and after AP staining, (b) Images of immunocytochemistry for pluripotency markers such as OCT4, SOX2, NANOG, TRA-1-81, TRA 1-60, SSEA-3 and SSEA-4. Blue indicates nuclei stained with Dapi (scale bar, 250 lm), (c) Generation of EBs using a fire finely bore glass pasteur pipette, (d) in vitro differentiation of ips cells into the three primary germ layers [Ectoderm TUJ1 (green), Endoderm-AFP (green) and FOXA2 (red), and Mesoderm-ASA (green) and SMA (red)], (e) Immunofluorescence analysis of teratoma sections after 60 days after intratesticular injection in SCID mice showing ectoderm [TUJ-1 and GFAP positive], endoderm [AFP and FOXA2 positive] and mesoderm [ASA and SMA positive] structures. over the patient s lifetime. These mutations are passed onto the ipscs during reprogramming and therefore promote the progressive loss of cellular function and cancer formation. In this contest, CB cells could minimize this problem since they are young carrying less somatic mutations. All these characteristics fortify CB as a very powerful and readily accessible cell source for the generation of healthy allogenic ipscs lines.
4 110 A. Giorgetti et al. The first report of ipscs induction from human blood cells utilized CD34+ mpb . However, compared to newborn CB stem cells, adult mpb cells will have the potential disadvantages that they may have accumulated genomic alterations as a result of ageing or disease, and that the pharmacological treatment used to mobilize the adult haematopoietic stem cell compartment represents a health risk for the donor . We have demonstrated that CB CD133+ cells, enriched in HSC and HPC, can be reprogrammed using 4 (OCT4, SOX2, KLf4 and c-myc, OSKM), 3 (OCT4, SOX2 and KLf4, OSK) as well as 2 (OCT4 and SOX2, OS) transcription factors. CB CD133+ cells were isolated using standard CD133 immuno-magnetic selection and infected by retroviral vectors. 15 days p.i. colonies, exhibiting typical hes cell morphology, appeared and we named them CB-iPSC. On average, infected CD133+ cells gave rise to 5 6 hes-like colonies and using independent CB units we could generate a total of 27 CB-iPSC lines. In addition, in order to exclude the possibility that the standard cryopreservation protocol did not affect the reprogramming ability, we have generated CB-iPSC lines from thawed CB units that had been stored frozen for more than 5 years. All the CB-iPSC lines were characterized for expression of pluripotency associated transcription factors and surface markers, and pluripotent differentiation ability in vitro and in vivo. CB-iPSC lines showed strong alkaline phosphatase and revealed expression of pluripotency markers such as OCT4, SOX2, TRA-1-81, TRA-1-60, SSEA3, SSEA4, and NANOG (Fig. 1a, b). All the CB-iPSCs tested could differentiate in vitro into derivatives of the three embryonic germ layers (Fig. 1c, d) and generate in vivo, upon injection into immuno-compromised SCID beige mice, complex intra-testicular teratomas (Fig. 1e). Additionally, following specific in vitro differentiation protocols, CB-iPSC gave rise also to specialized mesoderm-derived cell types such as rhythmically beating cardiomyocytes and ectodermal cells such as dopaminergic neurons. Finally, by global transcription profile and DNA promoter methylation analysis, we could demonstrate that CB cells are properly reprogrammed into pluripotent stem cells from both the expression and the epigenetic point of view. In addition, cytogenetic analysis showed that the CB-iPSC lines maintained a normal 46XY or 46 SPACC XX karyotype after more than 10 passages in culture. Moreover, the presence of male chromosomal excluded the possibility that the reprogrammed cells arise from a small fraction of contaminating mother cells known to be present in the initial CB sample. Our results confirmed that CB-iPSCs are transcriptionally reprogrammed to a state similar to hips and hes cells, are karyotypically stable, and show a differentiation potential consistent with pluripotency . Conclusion The possibility to generate ipscs from CB stem cells offer evident logistic advantages over the use of adult somatic cell types or adult stem cells for the purpose of creating ipsc cell banks. To date, more than CB units immunologically characterized are currently available worldwide through a network of CB banks [22,25]. This is the most comprehensive collection of potential source cells with diverse (and characterized) HLA types available, which could enable the generation of ipscs with perfect HLA match for any given patient. Moreover, selection of donors homozygous for common HLA haplotypes could be easily accomplished using banked CB units and would significantly reduce the number of CB-iPSC lines needed to provide a perfect HLA match for a large percentage of the population . However, although ipsc technology has advanced rapidly, it remains still unclear whether any ipscs will be therapeutically useful. Several technical issues, such as high efficiency, good differentiation protocol, functional engraftment and safety, have still to be improved and resolved before the full potential of ipscs can be realized. For example, recently it has been suggested that beyond the issue of whether the ipscs are derived with or without genomic integrations, the reprogramming process itself can produce mutations. Therefore, in order to have a realistic impact on a clinical level it would be useful set up protocols to generate better ipscs, as well as develop safety mechanisms to control those cells once they are transplanted. Disclosures The authors declare that there are no potential conflicts of interest. References 1 Thomson JA, Itskovitz-Eloor J, Smapiros S, et al.: Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: Reubinoff BE, Pera MF, Fong CY, et al.: Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000; 18: Nakajima F, Tokunaga K, Nakatsuji N: Human leukocyte antigen matching estimations in a hypothetical bank of human embryonic stem cell lines in the Japanese population for use in
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