Limits...
Making gametes from pluripotent stem cells--a promising role for very small embryonic-like stem cells.

Bhartiya D, Hinduja I, Patel H, Bhilawadikar R - Reprod. Biol. Endocrinol. (2014)

Bottom Line: However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints.These on further development produced sperm, oocytes and live offspring (had associated genetic problems).However, first the scientific community needs to arrive at a consensus about VSELs in the gonads and then work towards exploiting their potential.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Biology Department, National Institute for Research in Reproductive Health (ICMR), Mumbai 400 012, India. deepa.bhartiya@yahoo.in.

ABSTRACT
The urge to have one's own biological child supersedes any desire in life. Several options have been used to obtain gametes including pluripotent stem cells (embryonic ES and induced pluripotent iPS stem cells); gonadal stem cells (spermatogonial SSCs, ovarian OSCs stem cells), bone marrow, mesenchymal cells and fetal skin. However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints. A tremendous leap in the field occurred using mouse ES and iPS cells wherein they were first differentiated into epiblast-like cells and then primordial germ cell-like cells. These on further development produced sperm, oocytes and live offspring (had associated genetic problems). Evidently differentiating pluripotent stem cells into primordial germ cells (PGCs) remains a major bottleneck. Against this backdrop, we propose that a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) may serve as an alternative, potential source of autologus gametes, keeping in mind that they are indeed PGCs surviving in adult mammalian ovaries and testes. Both VSELs and PGCs are pluripotent, relatively quiescent because of epigenetic modifications of parentally imprinted genes loci like Igf2-H19 and KCNQ1p57, share several markers like Stella, Fragilis, Mvh, Dppa2, Dppa4, Sall4, Blimp1 and functional receptors. VSELs are localized in the basement membrane of seminiferous tubules in testis and in the ovary surface epithelium. Ovarian stem cells from mouse, rabbit, sheep, marmoset and humans (menopausal women and those with premature ovarian failure) spontaneously differentiate into oocyte-like structures in vitro with no additional requirement of growth factors. Thus a more pragmatic option to obtain autologus gametes may be the pluripotent VSELs and if we could manipulate them in vivo - existing ethical and epigenetic/genetic concerns associated with in vitro culture may also be minimized. The field of oncofertility may undergo a sea-change and existing strategies of cryopreservation of gametes and gonadal tissue for fertility preservation in cancer patients will necessitate a revision. However, first the scientific community needs to arrive at a consensus about VSELs in the gonads and then work towards exploiting their potential.

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Related in: MedlinePlus

Left yellow panel depicts event that occur naturally. Right purple panel represents human efforts to make synthetic gametes. Fertilization of gametes results in a blastocyst with inner cell mass (ICM) which comprises of pluripotent cells (grown in vitro as ES cells) and further develops into a epiblast-stage embryo where specification into somatic cells and primordial germ cells (PGCs) occurs. PGCs are pluripotent, express nuclear OCT-4, differentiate into gonocytes in testes and primordial follicles in ovaries (please refer to the main text for greater details) and persist in adult gonads as pluripotent, nuclear OCT-4 positive VSELs. Thus in addition to SSCs and OSCs in testes and ovaries [42], VSELs also exist [48] as reviewed recently. VSELs self-renew and give rise to progenitors (SSCs in testis and OSCs in ovary) which undergo clonal expansion, meiosis and further differentiation into gametes. Solid blue arrows represent asymmetric cell division of VSELs [48]. Differentiation of ES and iPS cells into synthetic gametes is a distant dream as they do not efficiently differentiate into PGCs. VSELs and OSCs spontaneously differentiate into oocyte-like structures in vitro[43, 63, 74–76, 78, 79] as they are indeed PGCs that survive into adulthood. Limited success has been achieved using bone marrow [27–29], fetal skin [30] and mesenchymal cells [31–33] possibly because they have VSELs present as a sub-group. Please note that brown color in the yellow panel represents pluripotent nuclear OCT-4 positive cells.
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Related In: Results  -  Collection

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Fig1: Left yellow panel depicts event that occur naturally. Right purple panel represents human efforts to make synthetic gametes. Fertilization of gametes results in a blastocyst with inner cell mass (ICM) which comprises of pluripotent cells (grown in vitro as ES cells) and further develops into a epiblast-stage embryo where specification into somatic cells and primordial germ cells (PGCs) occurs. PGCs are pluripotent, express nuclear OCT-4, differentiate into gonocytes in testes and primordial follicles in ovaries (please refer to the main text for greater details) and persist in adult gonads as pluripotent, nuclear OCT-4 positive VSELs. Thus in addition to SSCs and OSCs in testes and ovaries [42], VSELs also exist [48] as reviewed recently. VSELs self-renew and give rise to progenitors (SSCs in testis and OSCs in ovary) which undergo clonal expansion, meiosis and further differentiation into gametes. Solid blue arrows represent asymmetric cell division of VSELs [48]. Differentiation of ES and iPS cells into synthetic gametes is a distant dream as they do not efficiently differentiate into PGCs. VSELs and OSCs spontaneously differentiate into oocyte-like structures in vitro[43, 63, 74–76, 78, 79] as they are indeed PGCs that survive into adulthood. Limited success has been achieved using bone marrow [27–29], fetal skin [30] and mesenchymal cells [31–33] possibly because they have VSELs present as a sub-group. Please note that brown color in the yellow panel represents pluripotent nuclear OCT-4 positive cells.

Mentions: A careful review of published literature shows that a group from Japan, including Prof. Hayashi and Prof. Saitou has achieved major progress in the field of generating gametes from mouse pluripotent stem cells (mES/iPS cells). In 2011 they published in Cell that it is possible to obtain live pups from sperm derived from pluripotent stem cells (ES or iPS cells) [8]. In 2012 they published in Science that following a similar strategy, offspring are obtained from oocytes derived from ES or iPS cells [9]. In 2013, they have published their detailed protocols in Nature Protocols describing the method to generate eggs starting with mouse ES cells and iPS cells [10]. Basic reasoning that led to this remarkable success was that it is important to recapitulate in vitro what happens in vivo during early embryo development. Two main strategies that have been used in the past to induce germ cells from pluripotent stem cells (PSCs) include (i) spontaneous differentiation of PSCs to make embryoid bodies (EBs), isolate cells expressing germ cell markers for further manipulation and (ii) to use mouse epiblast stem cell lines to obtain germ cells. Both these approaches, although provide proof of concept that it may be possible to differentiate PSCs into germ cells, remain highly inefficient. Primordial germ cells (PGCs) are available in very few numbers and are relatively quiescent and thus the embryonic germ cell lines derived from them [11] have shown limited long-term proliferation potential [12]. Thus Hayashi’s group carried out experiments to first differentiate PSCs into epiblast-like cells and then induced them into PGC-like cells (PGCLCs). They demonstrate that once PGCLCs are obtained, it is possible to transplant them into testis/ovary to enable their further differentiation into sperm or oocytes respectively resulting in offspring. It is important to note that in both the publications, Hyashi et al. [8, 9] have reported existence of genetic anomalies in the offspring. When PSCs were induced to undergo spermatogenesis, some of the offspring underwent premature deaths because of tumors around the neck region. Similarly reduced number of pups were obtained from PSCs (3.9%) compared to those obtained by transplanting E12.5 PGCs (12.7%) or 3 weeks oocytes derived pups (17.3%). Almost half of the PSCs-derived oocytes failed to extrude second polar body resulting in 3PN zygotes. This is not surprising since extended cultures of ES/iPS cells are bound to result in the acquisition of genetic and epigenetic alterations during in vitro culture and parallel studies in humans remain a distant dream [2, 13]. Besides them, few other groups have also reported that PGCs have the ability to undergo gametogenesis when transplanted in adult tissues. Chuma et al. [14] transplanted PGCs in testis and obtained mature sperm whereas Matoba et al. [15] reported that PGCs isolated from E12.5 male fetus under the kidney capsule yield spermatids. Both these groups reported birth of healthy offspring. Similarly Matoba et al. [15] and Hashimoto et al. [16] reported that PGCs isolated from female fetus when transplanted under the ovarian bursa or kidney capsule result in functional eggs. It is intriguing to note that offspring born when starting with PGCs are normal compared to when starting with ES/iPS cells. Hayashi et al. [17] reviewed recent advances towards obtaining human gametes to treat infertility. They highlighted the existing hurdles in the existing differentiation protocols and discuss alternative use of germline stem cells (SSCs or OSCs) as a source to produce synthetic gametes (Figure 1). It may also be possible to obtain germ cells by transdifferentiation of somatic cells e.g. bone marrow and mesenchymal cells. Efforts are also ongoing to mature the primordial follicles in ovarian cortical tissue which are cryopreserved prior to cancer therapy.Figure 1


Making gametes from pluripotent stem cells--a promising role for very small embryonic-like stem cells.

Bhartiya D, Hinduja I, Patel H, Bhilawadikar R - Reprod. Biol. Endocrinol. (2014)

Left yellow panel depicts event that occur naturally. Right purple panel represents human efforts to make synthetic gametes. Fertilization of gametes results in a blastocyst with inner cell mass (ICM) which comprises of pluripotent cells (grown in vitro as ES cells) and further develops into a epiblast-stage embryo where specification into somatic cells and primordial germ cells (PGCs) occurs. PGCs are pluripotent, express nuclear OCT-4, differentiate into gonocytes in testes and primordial follicles in ovaries (please refer to the main text for greater details) and persist in adult gonads as pluripotent, nuclear OCT-4 positive VSELs. Thus in addition to SSCs and OSCs in testes and ovaries [42], VSELs also exist [48] as reviewed recently. VSELs self-renew and give rise to progenitors (SSCs in testis and OSCs in ovary) which undergo clonal expansion, meiosis and further differentiation into gametes. Solid blue arrows represent asymmetric cell division of VSELs [48]. Differentiation of ES and iPS cells into synthetic gametes is a distant dream as they do not efficiently differentiate into PGCs. VSELs and OSCs spontaneously differentiate into oocyte-like structures in vitro[43, 63, 74–76, 78, 79] as they are indeed PGCs that survive into adulthood. Limited success has been achieved using bone marrow [27–29], fetal skin [30] and mesenchymal cells [31–33] possibly because they have VSELs present as a sub-group. Please note that brown color in the yellow panel represents pluripotent nuclear OCT-4 positive cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4255929&req=5

Fig1: Left yellow panel depicts event that occur naturally. Right purple panel represents human efforts to make synthetic gametes. Fertilization of gametes results in a blastocyst with inner cell mass (ICM) which comprises of pluripotent cells (grown in vitro as ES cells) and further develops into a epiblast-stage embryo where specification into somatic cells and primordial germ cells (PGCs) occurs. PGCs are pluripotent, express nuclear OCT-4, differentiate into gonocytes in testes and primordial follicles in ovaries (please refer to the main text for greater details) and persist in adult gonads as pluripotent, nuclear OCT-4 positive VSELs. Thus in addition to SSCs and OSCs in testes and ovaries [42], VSELs also exist [48] as reviewed recently. VSELs self-renew and give rise to progenitors (SSCs in testis and OSCs in ovary) which undergo clonal expansion, meiosis and further differentiation into gametes. Solid blue arrows represent asymmetric cell division of VSELs [48]. Differentiation of ES and iPS cells into synthetic gametes is a distant dream as they do not efficiently differentiate into PGCs. VSELs and OSCs spontaneously differentiate into oocyte-like structures in vitro[43, 63, 74–76, 78, 79] as they are indeed PGCs that survive into adulthood. Limited success has been achieved using bone marrow [27–29], fetal skin [30] and mesenchymal cells [31–33] possibly because they have VSELs present as a sub-group. Please note that brown color in the yellow panel represents pluripotent nuclear OCT-4 positive cells.
Mentions: A careful review of published literature shows that a group from Japan, including Prof. Hayashi and Prof. Saitou has achieved major progress in the field of generating gametes from mouse pluripotent stem cells (mES/iPS cells). In 2011 they published in Cell that it is possible to obtain live pups from sperm derived from pluripotent stem cells (ES or iPS cells) [8]. In 2012 they published in Science that following a similar strategy, offspring are obtained from oocytes derived from ES or iPS cells [9]. In 2013, they have published their detailed protocols in Nature Protocols describing the method to generate eggs starting with mouse ES cells and iPS cells [10]. Basic reasoning that led to this remarkable success was that it is important to recapitulate in vitro what happens in vivo during early embryo development. Two main strategies that have been used in the past to induce germ cells from pluripotent stem cells (PSCs) include (i) spontaneous differentiation of PSCs to make embryoid bodies (EBs), isolate cells expressing germ cell markers for further manipulation and (ii) to use mouse epiblast stem cell lines to obtain germ cells. Both these approaches, although provide proof of concept that it may be possible to differentiate PSCs into germ cells, remain highly inefficient. Primordial germ cells (PGCs) are available in very few numbers and are relatively quiescent and thus the embryonic germ cell lines derived from them [11] have shown limited long-term proliferation potential [12]. Thus Hayashi’s group carried out experiments to first differentiate PSCs into epiblast-like cells and then induced them into PGC-like cells (PGCLCs). They demonstrate that once PGCLCs are obtained, it is possible to transplant them into testis/ovary to enable their further differentiation into sperm or oocytes respectively resulting in offspring. It is important to note that in both the publications, Hyashi et al. [8, 9] have reported existence of genetic anomalies in the offspring. When PSCs were induced to undergo spermatogenesis, some of the offspring underwent premature deaths because of tumors around the neck region. Similarly reduced number of pups were obtained from PSCs (3.9%) compared to those obtained by transplanting E12.5 PGCs (12.7%) or 3 weeks oocytes derived pups (17.3%). Almost half of the PSCs-derived oocytes failed to extrude second polar body resulting in 3PN zygotes. This is not surprising since extended cultures of ES/iPS cells are bound to result in the acquisition of genetic and epigenetic alterations during in vitro culture and parallel studies in humans remain a distant dream [2, 13]. Besides them, few other groups have also reported that PGCs have the ability to undergo gametogenesis when transplanted in adult tissues. Chuma et al. [14] transplanted PGCs in testis and obtained mature sperm whereas Matoba et al. [15] reported that PGCs isolated from E12.5 male fetus under the kidney capsule yield spermatids. Both these groups reported birth of healthy offspring. Similarly Matoba et al. [15] and Hashimoto et al. [16] reported that PGCs isolated from female fetus when transplanted under the ovarian bursa or kidney capsule result in functional eggs. It is intriguing to note that offspring born when starting with PGCs are normal compared to when starting with ES/iPS cells. Hayashi et al. [17] reviewed recent advances towards obtaining human gametes to treat infertility. They highlighted the existing hurdles in the existing differentiation protocols and discuss alternative use of germline stem cells (SSCs or OSCs) as a source to produce synthetic gametes (Figure 1). It may also be possible to obtain germ cells by transdifferentiation of somatic cells e.g. bone marrow and mesenchymal cells. Efforts are also ongoing to mature the primordial follicles in ovarian cortical tissue which are cryopreserved prior to cancer therapy.Figure 1

Bottom Line: However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints.These on further development produced sperm, oocytes and live offspring (had associated genetic problems).However, first the scientific community needs to arrive at a consensus about VSELs in the gonads and then work towards exploiting their potential.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Biology Department, National Institute for Research in Reproductive Health (ICMR), Mumbai 400 012, India. deepa.bhartiya@yahoo.in.

ABSTRACT
The urge to have one's own biological child supersedes any desire in life. Several options have been used to obtain gametes including pluripotent stem cells (embryonic ES and induced pluripotent iPS stem cells); gonadal stem cells (spermatogonial SSCs, ovarian OSCs stem cells), bone marrow, mesenchymal cells and fetal skin. However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints. A tremendous leap in the field occurred using mouse ES and iPS cells wherein they were first differentiated into epiblast-like cells and then primordial germ cell-like cells. These on further development produced sperm, oocytes and live offspring (had associated genetic problems). Evidently differentiating pluripotent stem cells into primordial germ cells (PGCs) remains a major bottleneck. Against this backdrop, we propose that a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) may serve as an alternative, potential source of autologus gametes, keeping in mind that they are indeed PGCs surviving in adult mammalian ovaries and testes. Both VSELs and PGCs are pluripotent, relatively quiescent because of epigenetic modifications of parentally imprinted genes loci like Igf2-H19 and KCNQ1p57, share several markers like Stella, Fragilis, Mvh, Dppa2, Dppa4, Sall4, Blimp1 and functional receptors. VSELs are localized in the basement membrane of seminiferous tubules in testis and in the ovary surface epithelium. Ovarian stem cells from mouse, rabbit, sheep, marmoset and humans (menopausal women and those with premature ovarian failure) spontaneously differentiate into oocyte-like structures in vitro with no additional requirement of growth factors. Thus a more pragmatic option to obtain autologus gametes may be the pluripotent VSELs and if we could manipulate them in vivo - existing ethical and epigenetic/genetic concerns associated with in vitro culture may also be minimized. The field of oncofertility may undergo a sea-change and existing strategies of cryopreservation of gametes and gonadal tissue for fertility preservation in cancer patients will necessitate a revision. However, first the scientific community needs to arrive at a consensus about VSELs in the gonads and then work towards exploiting their potential.

Show MeSH
Related in: MedlinePlus