Potential applications of germline cell-derived pluripotent stem cells in organ regeneration

Abstract

Impressive progress has been made since the turn of the century in the field of stem cells. Different types of stem cells have now been isolated from different types of tissues. Pluripotent stem cells are the most promising cell source for organ regeneration. One such cell type is the germline cell-derived pluripotent cell, which is derived from adult spermatogonial stem cells. The germline cell-derived pluripotent stem cells have been obtained from both human and mouse and, importantly, are adult stem cells with embryonic stem cell-like properties that do not require specific manipulations for pluripotency acquisition, hence bypassing problems related to induced pluripotent stem cells and embryonic stem cells. The germline cell-derived pluripotent stem cells have been induced to differentiate into cells deriving from the three germ layers and shown to be functional in vitro. This review will discuss the plasticity of the germline cell-derived pluripotent stem cells and their potential applications in human organ regeneration, with special emphasis on liver regeneration. Potential problems related to their use are also highlighted.

Introduction

Impressive progress has been made since the turn of the century in the field of stem cells and their potential for human regenerative medicine. Stem cells from varying sources are now moving into clinics. Due to the heterogeneity of diseases, as well as to the pathophysiology and anatomy of the targeted organs, there is still no stem cell that fits all requirements.

Embryonic stem (ES) cells, the gold standard for pluripotent stem cells, are derived from the inner cell mass of blastocysts. These cells show the most prominent amenability to in vitro expansion and have been depicted as a highly versatile donor source for basic research and experimental tissue repair.1 They have been shown to participate in chimera formation when microinjected into blastocysts and have been induced to differentiate into multiple cell types. However, ES cell-based therapy has been hampered by moral, legal and ethical dilemma surrounding the use of human embryos for derivation of ES. In addition, the nonautologous origin and tumorigenic potential of ES cells have prompted the finding of alternate sources of pluripotent stem cells that may have applications in human regenerative medicine.

Pluripotency has been conferred to adult cells by somatic cell nuclear transfer, parthenogenesis of unfertilized eggs, reprogramming by cell fusion and by forced expression of key transcription factors or use of small molecules.2–5 The team led by Yamanaka in 2006 first identified four transcription factors (Klf4, Sox2, Oct4 and c-Myc) that could reprogram mouse fibroblasts through retroviral transduction into pluripotent cells.6 This first generation of induced pluripotent stem (iPS) cells presented some concerns, as far as therapeutic value is concerned, due to the use of viral vectors as well as the potent oncogene c-Myc. A series of improvements thereafter followe,d and virus-free iPS cells can now be generated from human adult somatic cells, which brings the use of iPS cells a step closer to the clinic.7–9

Despite the potential of these iPS cells and their similarities to ES cells, significant obstacles to the clinical use of the former exist. The need to introduce transcription factors to induce pluripotency is an ongoing concern, especially if these iPS cells will have to be differentiated in vitro and undergo further modification, for instance, to correct genetic diseases. The technical hurdles concerning generation of iPS cells will surely be resolved in the near future, and the limitations associated with ES are not easily circumvented, hence the run for generating iPS cells from different cell types and with improved methods.10 The accelerated pace of iPS cell research has somewhat overshadowed the potentialities of other adult pluripotent stem cells, which do not require the above-mentioned manipulations to be reprogrammed.

One such cell type is the germline cell-derived pluripotent cell or GPSC (alias maGSC, mGS), which is derived from spermatogonial stem cells (SSCs). This review will discuss about the plasticity of the GPSCs and their potential applications in human organ regeneration, with special emphasis on liver regeneration. Potential problems related to their use are also highlighted.

The Spermatogonial Stem Cells

Spermatogenesis is a complex process involving a wide repertoire of cell types of the testis. It begins 5–7 days and 10–13 years after birth in rodents and humans, respectively, and continues throughout life, hence requiring the continuous presence of a small pool of SSCs. Testicular SSCs attached to the basement membrane of the seminiferous tubules are unipotent and continuously generate differentiating daughter cells for subsequent production of haploid spermatozoa through meiosis.11 Surrounding Sertoli cells provide the necessary architectural support as well as the essential factors for the maintenance of this rare population of stem cells (usually 1 in 3,000 testicular cells). SSCs, despite the absence of specific markers, have been isolated from mouse testis and successfully cultured in vitro for several months. The only way of identifying an SSC thus far is by cell transplant into the seminiferous tubules of infertile recipient mice, as first described by Brinster et al.12,13 Human SSCs have also been isolated and long-term cultures established.14,15 The initial attempts to culture human SSCs in vitro for more than a few passages were unsuccessful probably due to contaminating somatic cells.16 Enrichment of human SSCs using cell surface markers like CD9, GPR125 or integrin α6 allowed cultures of these cells for extended period of time (over six months).14,15,17 Cryopreserving human SSCs, especially before the start of any cancer therapy, is becoming a necessity. Recently, Sadri-Ardekani et al. demonstrated that testicular cells from six males undergoing orchiectomy for prostrate cancer treatment contained SSCs.18 These SSCs were isolated and cultured for up to 15 weeks. The ability to provide long-term culture and propagation of SSCs from humans in vitro is a remarkable leap towards clinical applications.19 SSC culture may also help in understanding human spermatogonial renewal mechanisms.

Once removed from their niche and cultured in vitro, SSCs acquire a larger developmental potential than in vivo. It appears that SSCs can directly transdifferentiate into other cell types when combined with the appropriate mesenchyme.19 SSCs have been injected directly into the renal parenchyma of mature female mice, and fluorescence in situ hybridization technology was used to analyze which types of cells differentiated from SSCs. Interestingly, the Y chrosomome was evidenced in the nucleolus of epithelial cells of the renal tubules as well as podocytes of the glomerulus showing direct differentiation of SSCs into renal cells in vivo.20 However, cell fusion was not verified by these authors, and no in vitro demonstration of differentiation towards this specific lineage exists.

Neonatal mouse SSCs were also shown to directly differentiate into prostatic, uterine and skin epithelium, accompanied by the expression of respective epithelial proteins when combined with the appropriate fetal or neonatal mesenchyme and grafted in vivo.21 It has also been demonstrated that adult testicular cells, isolated from seminiferous tubules, mixed with limiting dilutions of dispersed mammary epithelial cells and injected into epithelium-divested mammary fat pads could adopt mammary epithelial progenitor cell properties, including self-renewal and the production of cell progeny, which differentiate into functional mammary epithelial cells.22 This potential plasticity of SSCs needs to be investigated in more depth.

Germline Cell-Derived Pluripotent Stem Cells

Since the pioneer work by Kanatsu-Shinohara et al., who demonstrated that ES-like cells could be derived from SSCs in long-term in vitro cultures, ardent research has been ongoing in several laboratories to render the use of these cells a valid clinical option.23 Cultured SSCs derived from neonatal testis undergo a spontaneous conversion at low efficiency after 4–7 weeks into ES-like pluripotent stem cells; hence the term germline cell-derived pluripotent stem cells or GPSCs.23–25 The phenomenon of conversion of germline cells to pluripotent ES-like stem cells upon culture stimulation was first observed for cells isolated from the mouse embryo (primordial germ cells) and neonatal testis but has now been successfully reproduced using SSCs from the adult.24–26 GPSCs, which share features of both ES cells and adult stem cells, are of prime importance in tissue regeneration. GPSCs express pluripotent markers, such as Oct3/4, Sox2 and Nanog, and are phenotypically similar to ES cells. Moreover, they produce teratomas when inoculated into mice and form germline chimeras when microinjected into mice blastocysts. GPSCs and ES cells also have similar miRNA profiles.27 However, GPSCs differ from ES cells in their genomic imprinting pattern and in limited gene expression profile due to the differing origin of these two cell types.28,29

Isolation and culture of SSCs and derivation of GPSCs have now been successfully reproduced in humans (termed herein hGPSCs to distinguish these from mouse GPSCs).14 These hGPSCs are similar to human ES cells at cellular and molecular levels and have been continuously passaged for up to 16 months, over more than 40 passages, without decline in colony-forming ability. They could be cryopreserved and thawed with no loss in proliferation or differentiation capacity.17,30,31 However, there is discrepancy between the reports of Conrad et al. and Kossack et al., mainly due to the SSC isolation methods employed.17,30 The former used a three-step procedure to isolate integrin α6⁺ SSCs from human testicular parenchymas and derived pluripotent ES-like cells (22 out of 22 patients) while the latter cultured testicular cells from testis biopsies using human ES media with subsequent transfer onto mouse embryonic fibroblasts and derived ES-like cells (1 patient) that differed from human ES cells in gene expression, methylation and ability to form teratomas and which were thus considered multipotent. Other groups independently corroborated the above-mentioned reports describing culture of human SSCs and derivation of hGPSCs. Golestaneh et al. obtained pluripotent hGPSCs, which formed the three germ layers upon EBs formation and formed teratoma when injected in nude mice.32 Mizrak et al. obtained multipotent hGPSCs, which did not form extensive teratomas upon injection into immuno-compromized mice.31 Improved and standardized SSC isolation and culture methods need to be established to clarify this issue of pluripotency/multipotency of hGPSCs. Moreover, the identity of the human germ cells, collectively termed spermatogonia or SSCs, that convert to ES-like cells should be better characterized. Importantly, as opposed to iPS cells, the starting cell type for GPSCs is already poised to adopt a pluripotent stem cell fate when stimulated in specific culture conditions. This is indicated by the spontaneous generation of GPSCs from SSCs, without genetic manipulation of the cells. Idealistically, hGPSCs may be generated from SSC cultures and stored in a cell bank after characterization (histocompatibility, donor information, etc.), for differentiation in specific cell types for transplantation. One of the drawbacks is the low efficiency of formation of ES-like cells from SSC cultures in current in vitro conditions. Research on functional small molecules facilitating GPSC generation may help in achieving a solution.33,34 Thus, SSCs, and especially hGPSCs, could become very important tools for human cell-based gene therapy and solicit considerable interest in investigating their differentiative capacity. The attempts at differentiating GPSCs into specific cell types and their potentiality for in vivo use are summarized hereafter.

GPSCs and Liver Regeneration

Liver diseases, in particular chronic hepatitis, commonly caused by hepatitis C (HCV) and B viruses, alcoholism, nonalcoholic fatty liver disease, autoimmune and drug-induced hepatic disorders, affect hundreds of millions of patients worldwide. Since the potential progression from chronic hepatitis to cirrhosis, liver failure and hepatocellular carcinoma is well known, the efficacy of preventing and treating these causative agents is being studied.34

With advances in both surgical technique and immunosuppression regimen, orthotopic liver transplantation (OLT) has become the standard of care for the treatment of patients with end-stage liver disease, and, as a result, the demand for OLT now far exceeds the current donor organ supply. The ongoing organ shortage has reached a critical magnitude, highlighting the burden of recurrent incurable disease. For example, although OLT is not a cure for HCV infection, HCV-related cirrhosis has become the most frequent indication for OLT, with around 50% of liver transplant recipients carrying this diagnosis (43% between 1990–2002 in Turin, Italy).35 Importantly, hepatocytes retain their replicative capacity, unlike other cell types. The liver tissue mass can be restored after two-thirds hepatectomy, as shown by Higgins and Anderson in the 1930s in rat models.36 The human liver also contains a population of stem cells as well as bipotential oval cells that can differentiate into hepatocytes when necessary.37,38 Unfortunately, in severe liver diseases, the hepatocytes' and the liver stem cells' regenerative capacity may be impaired. Thus, much hope of curing the diseased liver has turned towards cell therapy.

The latter has numerous potential advantages when compared to OLT, since transplantable cells can be expanded in vitro and cryopreserved, thus abolishing the limit of organ shortage; can be genetically manipulated to correct inborn errors of metabolism; cryopreserved for future use; infused without major surgery and obtained from the same patient, hence avoiding risk of rejection and need for lifelong immunosuppression.39

Current approaches to cell therapy in humans have involved the use of an extracorporeal liver support system based on primary human hepatocytes and albumin dialysis.40 In other studies, cell transplants in humans have been attempted by the use of human hepatocytes isolated from cadaveric livers.41 However, donor organs are scarce, and the need for lifelong immunosuppressive therapy, as well as the heterogeneity of patients, lack of controls and follow-up duration, greatly limit this approach.

Hepatocyte-like cells have been derived by directed differentiation from bone marrow, adipocytes and mesenchymal stem cells.42 However, when these cells are cryopreserved, a loss in viability is observed.43,44 ES cells have hitherto been the most promising cell source for hepatocyte generation. Both mouse and human ES cells have been used for this purpose.45–47 However, ES cells are allogenic in nature, and transplantation of hepatocyte-like cells generated from them will require immunosuppression, in addition to the other issues described above. Thus, it has become imperative to find alternative sources of cells for liver repair.

Several groups have reported the expression of the early hepatic marker, a fetoprotein, in embryoid bodies (EBs) generated from mouse GPSCs. The directed differentiation of testis-derived pluripotent stem cells into hepatocyte-like cells was also reported, but the characterization of the latter at functional level was not performed.48 We showed, for the first time, that metabolically active hepatocytes, capable of albumin and haptoglobin secretion, urea synthesis, glycogen storage and indocyanine green uptake can be derived from GPSCs in vitro.29 In this study, a large-scale microarray analysis was conducted to compare GPSCs to ES cells during hepatocyte differentiation, and a marked similarity in the gene expression profile between these two cell lines was evidenced. The GPSC-derived hepatocytes were found to closer to fetal hepatocytes (embryonic day 16) than adult hepatocytes (post natal day 1). The in vivo colonization capacity of these cells in mouse models of liver diseases remains to be investigated.

GPSCs are thus a promising tool for the treatment of liver diseases whilst avoiding ethical concerns over embryo use for the production of human pluripotent stem cells. In addition, GPSC-derived hepatocytes may provide the necessary foundation for cell-based therapy for metabolic diseases currently treated by OLT, especially those that can be corrected by only a small amount of functional protein. Hepatocytes derived from GPSCs may prove to be a renewable source of transplantable organs, overcoming the current organ shortage. These cells may also serve as “bridging therapy” between disease diagnosis and organ transplant, as in the case of newborns with genetic defects who have to reach the right age for surgical operation. Moreover, liver transplant mainly requires blood group compatibility, thus bypassing the problem of “perfect” match between donors and recipients.49 In this case, both males and females may benefit from the GPSC-derived hepatocytes. Other advantages of using GPSCs are that these cells can be passaged more than 30 times without changes in karyotype, and that they can be frozen and thawed, much like ES cells. Furthermore, generating and establishing these cell lines as well as making them differentiate into hepatocytes currently takes nearly 6 months (approximately 5 months for culture of SSCs and derivation of GPSCs and 1 month for differentiation of GPSCs into hepatocytes), which, though seemingly a long time, assuming translation to the clinical setting, is far less than the time the patient has to wait when on the transplant list.

GPSCs and Heart Regeneration

Cardiovascular disease remains the leading cause of morbidity and mortality in the western world.50 Heart transplantation is one the most effective treatment for cardiac failure or other cardiomyopathies. The solution for organ scarcity may be offered by cell therapy. Increasing evidence indicates that the mammalian heart contains a population of tissue-specific progenitor cells capable of differentiating into cardiomyocytes, smooth muscle cells and endothelial cells upon request. The heart can also be repopulated by cells from extracardiac sources. Several cell types are being studied as potential sources of cardiomyocytes for cell transplantation therapy and tissue repair in various cardiomyopathy models. Transplantable cell sources identified to date include cardiac progenitor cells,51 fetal cardiomyocytes, mesenchymal stem cells,52 bone marrow cells, ES cells,53 iPS cells54 and very small embryonic-like cells (VSELS).55 So far, clinical trials on cardiac regeneration involving stem cells have been moderately successful, and thus, it remains unclear which candidate is best for cell transplantation therapy. The inadequacy of these cells to restore the cardiac function of a failing heart has incited the search for alternative therapeutic approaches for substituting lost cardiomyocytes with new functional ones as an alternative to heart transplant. Pluripotent stem cells like GPSCs could offer a substitute to ES cells in cardiomyocytes generation. An interesting study carried out by Guan et al. reported for the first time that GPSCs can be induced to differentiate into functional cardiomyocytes.56 Through molecular, cellular and physiological assays, they demonstrated that GPSC-derived cardiomyocytes had similar properties to those derived from ES cells. Transplantation of GPSCs into the left ventricular free wall of female mice showed that these cells engrafted and were present in heart sections one month post-injection. These cells were able to proliferate and differentiate in the normal heart without teratoma formation. However, the GPSCs differentiated in loco into vascular endothelial and smooth muscle cells. No spontaneous in vivo differentiation into cardiomyocytes were observed in the normal heart. This was probably due to the fact that the local microenvironment is crucial in determining the fate of engrafted cells. Cytokines or growth factors released during myocardial infarction might have promoted the correct differentiation of GPSCs into cardiomyocytes in vivo. Differentiating GPSCs into cardiomyocytes in vitro prior to intramyocardial injection into mice necessitating cardiac regeneration may also circumvent this problem. Another study, carried out by Baba et al., showed that Flk1⁺ cells from differentiating GPSCs could give rise to mature cardiomyocytes and endothelial cells as efficiently as ES cells.57 The same group also demonstrated that transplantation of Flk1⁺ GPSCs directly into the heart of ischemic mice improved cardiac function.58 Although 4 weeks after treatment, the number of cardiomyocytes derived from Flk1⁺ GPSCs were too small to account for the improvement in cardiac function, angiogenesis around ischemic area was enhanced compared to control group, and senescence was also remarkably diminished in the early phase of ischemia.58 The present findings open new possibilities for basic research on cardiac development as well as cardiac regeneration.

GPSCs and Brain Regeneration

The notion of cell replacement therapy to treat neurodegenerative diseases is not new. There are now over two decades of experimental evidence that fetal cell transplantation can be an effective strategy for cellular repair and functional recovery in animal models of neurodegenerative diseases.59 However, the limited availability of fetal tissues, combined with the variable clinical outcomes observed to date, has prompted the search for alternative sources of donor cells for therapeutic intervention. A wide range of stem cells is currently being investigated for cell therapy of neurodegenerative diseases. ES cells have great propensity to form neural precursors, mature neurons and glia upon induction in vitro. It has been shown that dopaminergic neurons from both mouse and human ES cells are capable of integrating host tissue in rodents with Parkinson disease.60–62 Neural stem cell-like populations have been also generated from adult somatic stem cells, like adult human bone marrow stromal cells or skin fibroblasts.63–65 Other studies reported that bone marrow stem cells transplanted into the mouse forebrain after focal occlusion of the middle cerebral artery, an experimental model of ischemic stroke, largely relocate to the infarct penumbra, undergo a process of in situ proliferation and develop into putative cortical interneurons during the early phase of experimental vascular injury.66 GPSCs also show great promise for the generation of neural cell types. Streckfuss-Bomeke et al. have shown that neural progenitors can be obtained from GPSCs under specific culture conditions.59 These progenitors further matured into functional neurons (GABAergic, glutamatergic, serotonergic and dopaminergic neurons) and glial cells (astrocytes and oligodendrocytes). Electrophysiological recordings of passive and active membrane properties and postsynaptic currents demonstrated the maturation of GPSC-derived precursor cells into functional cells.59 Glaser et al. also showed that GPSC-derived neurons generate active functional networks, which use both GABAergic synaptic transmission and engage in synchronized oscillatory activity.1 Another interesting observation was that GPSC-derived oligodendrocytes undergo full maturation and ensheathe host axons in myelin-deficient tissue in organotypic slice cultures of the myelin-deficient rat cerebellum. GPSC-derived neural cells thus constitute a promising cell source for the treatment of many different nervous system disorders. Although GPSC-derived neurons are functional in vitro, their potential to engraft into the damaged host brain and function as neurons after transplantation remains to be demonstrated. Besides their potential for autologous cell therapy, the neural differentiation of GPSCs opens a new window for basic research on neural development and neural regeneration.

GPSCs and Hematopoietic Potential

Hematopoietic stem cell (HSC) transplantation has been widely used for treating hematological malignancies and inherited disorders. Peripheral blood and cord blood stem cells as well as bone marrow cells have been intensively studied and shown to be effective for clinical use. Recently, ES cells have been proposed as an alternative candidate source of HSCs. However, due to the problems associated with transplantation of ES-derived cells, attempts to derive hematopoietic cells from GPSCs have been made. GPSCs have been shown to differentiate, on OP9 cells, into CD45⁺ hematopoietic cells, including Gr-1⁺Mac1⁺ myeloid cells and Ter119⁺ erythroid cells.23 In a recent report, Yoshimoto et al. reported the generation of multipotent hematopoietic progenitor cells with myeloid and lymphoid potential emerging from FLK1⁺-differentiating GPSCs using the OP9 feeder cell system and described the localization of GPSC-derived hematopoietic cells in the bone marrow cavity after intra-bone marrow injection in immunodeficient mice.67 However, the GPSC-derived hematopoietic cells could engraft in the host but did not proliferate or display multilineage repopulating ability in vivo. Thus, much optimization needs to be done in the field of hematopoietic research, but these data serve as a prelude to more promising results in the future.

Potentialities of Human GPSCs

Differentiation studies on hGPSCs are still in their early days, but it has been demonstrated that hGPSCs are capable of differentiating in vitro into derivatives of all three germ layers. Conrad et al. examined teratoma formation efficiency of hGPSCs derived from eight patients by injecting cells intramuscularly or subcutaneously in the neck region of adult nude mice.17 Teratomas that formed after 6–8 weeks contained foci with derivatives of endodermal, mesodermal and ectodermal germ layers, which was confirmed by detailed analysis of tissue sections. These authors also showed that hGPSCs can be differentiated morphologically in vitro into myogenic, osteogenic, pancreatic and neural lineages. Moreover, the hGPSC-derived pancreatic cells also produced insulin-, glucagon- and c-peptide-positive cells. The independent hGPSC cell line generated by Kossack et al. also gave rise spontaneously to the three germ layers upon EBs formation.30 However, no extensive teratoma formation was found when these cells were injected under the kidney capsule of SCID mice. This discrepancy in teratoma formation by hGPSCs between these two studies may be due to the differing cell injection sites and methods used.68 Human GPSCs can also be induced to differentiate into NES⁺, MAP2⁺ and TUBIII⁺ neural linages after 6 weeks' culture in neural cell-specific media.30 Moreover, hGPSCs were also induced to differentiating into pancreatic β cells, which were shown to produce insulin in response to glucose and osteogenic-differentiated cells that showed deposition of phosphate and calcium, demonstrating their functional capacity.31 The hGPSCs are thus extremely promising, and their full potentiality still needs to be investigated, first, at the preclinical level in vivo in mouse models, then in clinical trials on humans.

Conclusions

The discovery of adult GPSCs and their differentiation plasticity offers great promise for human regenerative medicine whilst avoiding ethical concerns over embryo use for production of pluripotent human stem cells. And, most importantly, no viral vectors or special manipulations are required for “reprogramming” these adult stem cells into pluripotent stem cells.

There is good reason to think that GPSCs, due to their ES cell-like plasticity, can be induced to differentiate into cell types other than those described above (summarized in Fig. 1) under specific culture conditions.

There are, however, several concerns that need to be addressed before a clinical trial with hGPSC-derived cells can be initiated. First, human SSC isolation and hGPSC derivation methods need to be standardized; second, the differentiation capacity of hGPSCs needs to be fully investigated; third, participation of GPSCs in tissue regeneration has to be correctly assessed in vivo in rodents; fourth, being uniparental, GPSCs have silenced H19 imprinted gene on both alleles. H19 is a tumor suppressor gene, and alteration in its expression is involved in the pathogenesis of some human tumors.69 Finally, more progress is needed in terms of allogenic hGPSC transplantation.

Assuming translation of the SSC/GPSC system to humans, the conversion of cultured adult SSCs into cells with pluripotent features holds great therapeutic potential for degenerative diseases. The availability of compatible, pluripotent cells for transplantation from simple biopsies would circumvent immunological problems associated with regular human ES cell-based technology.

However, like ES and iPS cells, these GPSCs are target of lymphokine-activated killer (LAK) cells and natural killer (NK) cells in vitro. Moreover, NK cells were able to kill these pluripotent stem cells in vivo too.70 Interestingly, differentiation induced resistance to NK and LAK cells, indicating the importance of eliminating residual pluripotent stem cells from differentiated ones before cell transplant can be considered.

In conclusion, GPSCs may offer a safer means of generating pluripotent cells than other adult cells and provide new opportunities for getting patient-specific regenerative therapy. Cell therapy with hGPSCs will surely bring great improvements over the current organ regeneration strategies mentioned above. An “ethical” stem cell has probably been found.

Key Messages

• As an alternative to ES cell-based therapy, which has been hampered by moral, legal and ethical dilemma, there are the germline cell-derived pluripotent cells or GPSCs, which are ES-like cells derived from spermatogonial stem cells.

• GPSCs derived from mice and humans are naturally pluripotent/multipotent, and share features of both ES cells and adult stem cells, hence rendering them very important in tissue regeneration.

• Metabolically active hepatocytes, capable of albumin and haptoglobin secretion, urea synthesis, glycogen storage and indocyanine green uptake have been derived from GPSCs in vitro and may be used for cell therapy of the liver.

• Other promising applications of GPSCs have been reported for heart and brain regeneration and in hematology.

Abbreviations

EBs	=	embryoid bodies
ES	=	embryonic stem
GPSCs	=	germline cell-derived pluripotent stem cells
hGPSCs	=	human GPSCs
iPS	=	induced pluripotent stem
LAK	=	lymphokine-activated killer
MaGSCs	=	multipotent adult germline stem cells
mGS	=	multipotent germline stem
NK	=	natural killer
OLT	=	orthotopic liver transplantation
SSC	=	spermatogonial stem cells
VSELS	=	very small embryonic-like cells

Figures and Tables

Figure 1 Differentiative potential of GPSCs. SSCs can be cryopreserved for restoring fertility in patients undergoing chemotherapy. When cultured, SSCs give rise to ES-like colonies (GPSCs) that can be induced to differentiate into cells of different lineages. Hepatocytes generated from GPSCs are functional. A PAS-positive hepatocyte is shown.

Acknowledgments

Our studies were supported by grants from PI-STEM (Regione Piemonte), PRIN 2008. Special thanks to Letizia De Chiara and Francesca Barbieri for experimental help.