Background
Alternate sources of stem cells to make gametes in vitro
Making sperm in a dish
Differentiation of hES/iPS cells into male germ cells/sperm in vitro
Differentiation of VSELs into male germ cells/sperm in vitro
Other approaches to obtain sperm in vitro
On the crossroads to treat male infertility
Study | Highlights |
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Vahdati et al. [66] | The study evaluated the regenerative effect of bone marrow derived stem cells (CD29+/CD73+/CD45-) on spermatogenesis of busulphan treated infertile hamster. Following 60 days after efferent duct injection of bone marrow stem cells, histological evaluation of testis showed presence ofspermatogonia, primary spermatocytes, spermatids and sperm in seminiferous tubules compared to negative controls. |
Maghen et al. [67] | The study evaluated the role of human umbilical cord blood MSCs in regeneration of testicular niche. In addition to supporting the expression of murine germ cells and putative SSCs in vitro, the in vivo potential was evaluated by transplantation of MSCs into azoospermic mouse model. Transplanted cells majorly localized into interstitial space. Following transplantation, partial reconstruction of seminiferous tubule architecture was observed. |
Abd Allah et al. [68] | The study evaluated the effect of transplantation of human cord blood derived mesenchymal cells (CD34-) and hematopoietic stem cells (CD34+) by local injection into testis of busulphan treated recipient mice. Testicular histoarchitecture was found normal and sperm were present in lumen in mesenchymal cells transplanted group compared to the HSC transplanted group. l |
Ghasemzadeh-Hasankolaei et al. [69] | The study evaluated the fate of autologous bone marrow MSCs (isolated and labelled with PKH26) after transplantation into testes of busulphan treated Wistar rats. Transplanted bone marrow MSCs were studied at 3 time points (4,6 and 8 weeks) after transplantationand were found to survive post transplantation as studied by PKH26 expression. Some transplanted cells homed at germinal epithelium and expressed germ cell markers DAZLand STELLAindicating differentiation to spermatogonia. |
Rahmanifar et al. [70] | The study aimed to evaluate the seminiferous tubules of azoospermic rats following bone marrow derived MSCs transplantation. The recipient mice were prepared by busulphan treatment. Following efferent duct injection of MSCs, the transplanted tubules showed spermatogenesis with presence of germinal cells like spermatogonia, primary spermatocytes, spermatids and sperm. |
The study showed that VSELs survive busulphan treatment in the testis and resume spermatogenesis when mesenchymal/Sertoli cells are transplanted through the intertubular route. | |
Chen et al. [71] | In vivo differentiation potential of human cord blood mesenchymal stem cells was evaluated following transplantation into busulphan treated mice seminiferous tubules. Transplanted tubules exhibited improved histology compared to busulphan treated tubules. |
Yang et al. [72 | The study investigated the potential of human umbilical cord MSCs (CD31−/CD73+/CD105+) to promote spermatogenesis regeneration in busulphan treated testis following interstitial injections. Three weeks after injection, there was an increased expression of meiotic markers namely, Dazl, Vasa, Stra8, Scp3, Cyclin A1, Tnp2, Pgk2, Miwi, Tex18 and Akap3. Protein level expression of MIWI, VASAa and SCPwas also increased compared to controls. |
Sabbaghi et al. [73] | Rat bone marrow MSCs (5-10 × 106 cells) were cultured and transplanted via rete testis into torsioned azoospermic testis. Germ cell specific markers (OCT4, VASAand c-KIT) were monitored for the differentiation of MSCs after transplantation. |
Aziz et al. [74] | Bone marrow derived MSCs were transplanted into busulphan treated rats. Results showed that MSCs have potential for in vivo transdifferentiation into spermatids and spermatocytes. |
Making oocytes in a dish
Differentiation of hES/iPS cells into female germ cells/oocytes in vitro
Differentiation of VSELs into female germ cells/oocyte-like structures in vitro
Study | Highlights |
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Wang et al. [107] | Menstrual blood mono-nuclear cells were transplanted via intra-peritoneal route into mice chemoablated with cisplatin. The transplanted cells localized into ovarian interstitium. Following transplantation, follicle numbers increased and levels of sex hormones reached normalcy and improved ovarian function. |
Fouad et al. [108] | The study compared the efficiency of human amniotic membrane and adipose derived MSCs following transplantation into cyclophosphamide induced ovarian failure. Transplantation of MSCs from both sources showed increased number of follicles and oocytes alongwith increase in serum estradiol and decrease in serum FSH compared to chemoablated controls. The efficacy was more using human amniotic membrane MSCs. |
Song et al. [109] | In cyclophosphamide induced POF rat model human umbilical cord MSCs were transplanted by using either tail intravenous injection or injection into ovary in situ. Following transplantation, folliculogenesis was recovered along with hormonal secretions and decreased ovarian cell apoptosis. |
Kilic et al. [110] | Transplantation of bone marrow MSCs into cyclophosphamide treated rat showed protective effects by reducing germ cell apoptosis and DNA damage. Increased primordial follicular counts were obtained following transplantation compared to controls. |
Liu et al. [111] | Bone marrow derived MSCs were transplanted into POF rat model obtained by cisplatin treatment. The transplanted group showed increase in follicle growth and estradiol levels compared to control, thereby restoring ovarian structure and function. |
Liu et al. [112] | Transplantation of human endometrial MSCs into POF mouse model induced by cyclophosphamide showed survival of transplanted cells in ovaries and upregulation of ovarian markers along with increased estradiol and follicle number compared to control and restoring ovarian function. |
Lai et al. [113] | Following transplantation of skin derived MSCs from male/female mice into recipient mice with busulphan and cyclophosphamide induced ovarian damage, partial restoration of fertility was observed. Transplanted MSCs grafted into host ovaries and increased expression of oogenesis markers was observed compared to controls. |
Abd Allah et al. [114] | MSCs from bone marrow of male rabbits were injected intravenously into female rabbits chemoablated with cyclophosphamide. Increase in follicle numbers and resumption of normal follicular structure was observed compared to controls by histology. Ovarian tissues showed presence of Y-chromosome containing donor cells indicating engrafting of transplanted cells. |
Wang et al. [115] | Umbilical cord MSCs were transplanted intravenously into POF mouse model made by cyclophosphamide treatment. Reduced apoptosis in cumulus cells, increased number of follicles and recovery of ovarian function was observed. |
Liu et al. [116] | Transplantation of human amniotic fluid cells (CD44+/CD105+) showed survival into cyclophosphamide induced POF mice models for atleast 3 weeks following transplantation and proliferated. |
Selesniemi et al. [117] | Bone marrow mono-nuclear cells from young adult female mice (with EGFP transgene under β-actin) were transplanted into young/middle aged females. Following this treatment, the fertile potential of the aging female was sustained for longer period than the normal reproductive senescence. Offsprings did not show EGFP expression. However, donor bone marrow derived somatic cells accumulate in recipients indicating efficient donor cell engraftment. |
Fu et al. [118] | Effect of MSC transplantation on ovarian damage induced by chemotherapy using cyclophosphamide in rats was studied. Cultured MSCs were labelled with GFP and transplanted directly into bilateral ovaries. Following transplantation, the ovarian function was improved. Reduced germ cell apoptosis and upregulation of Bcl-2 was found in vivo. |
Other approaches to obtain oocyte-like structures in vitro
On the crossroads to treat female infertility
Obtaining gametes from non-gonadal sources
References | Extra-gonadal sources from which gametes have been obtained |
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Female | |
Lai et al. [122] | Human Menstrual blood |
Ge et al. [123] | Human skin cells |
Yu et al. [124] | Human amniotic fluid |
Dyce et al. [125] | Porcine fetal skin |
Lee et al. [126] | Mouse BM and PB |
Dyce et al. [127] | Clonal pancreatic stem cell line |
Dyce et al. [128] | Fetal and new born porcine/mouse/human skin |
Johnson et al. [129] | Mouse bone marrow and peripheral blood |
Male | |
Shirazi et al. [23] | Adult mouse bone marrow |
Shaikh et al. [22] | Adult mouse bone marrow |
Hua et al. [130] | Human fetal bone marrow |
Drusenheimer et al. [131] | Human bone marrow |
Nayernia et al. [40] | Mouse bone marrow |
Discussion and conclusions
Various approaches | Current status |
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Making Artificial Gametes from ES/iPS Cells | • Protocols are not yet available to convert human ES/iPS cells into PGCLCs. Obtaining gametes from mouse ES/iPS cells is successful but still inefficient and has associated epigenetic concerns. • Human ES cells exist in primed state and have to be converted to naïve state for further differentiation • Gametes obtained from human ES cells will not ensure biological parenthood unless and until the hES cells are derived by somatic cell nuclear transfer (therapeutic cloning). However, despite recent success in the field [130], this may also not be a practical solution since the hES cells derived by this method will have to be first converted into PGCLCs which is a road-block at present. • Serious concern exists with the use of iPS cells to make gametes. They exhibit genomic [131, 132] and mitochondrial [133] DNA mutations which seriously limit their clinical utility [134]. They also retain residual epigenetic memory of the somatic cell from which they are derived [135, 136]. Even for other clinical conditions, it is being advocated to use allogeneic over autologus iPS cells as it is more practical and safe. The first clinical trial for macular degeneration using autologus iPS cells was suspended due to safety concerns [137]. But use of allogeneic iPS cells to make gametes will not ensure biological parenthood. • Gametes obtained from hES/iPS cells will require use of assisted reproductive technology to have a baby. But this approach is expensive and inefficient. |
Restoring Fertility by Targeting Endogenous, Resident Stem Cells (VSELs) | • Major advantage of VSELs compared to hES/iPS cells is that they are equivalent to PGCs. Developmentally they are obtained from epiblast-stage embryo and thus relatively more mature compared to ES cells obtained from the inner cell mass of blastocyst stage embryo. • Being equivalent to PGCs (natural precursors to gametes), VSELs spontaneously differentiate into sperm & oocytes in vitro • VSELs survive oncotherapy in the gonads and transplanting niche cells (autologus mesenchymal cells from any source) can regenerate non-functional, azoospermic testis and POF ovary. This approach will ensure restoration of normal fertility. If mesenchymal cells are transplanted in pediatric cancer survivors, the gonads could serve as a source of hormones for secondary sexual development and later on in life will also be a source of gametes. • This approach could circumvent all associated safety and epigenetic concerns that invariably creep in when cells are cultured in vitro. Normal fertility will be restored and there will be no need of use of assisted reproductive technology. This approach is more feasible and less expensive. Most importantly, this approach could obviate the need to cryopreserve gonadal tissue prior to oncotherapy. |
Other Available Options | • All the below mentioned alternatives are still being researched upon and are not yet ready for the clinics • In vitro follicle culture • Artificial ovary • Entire ovary cryopreservation • Use of feto-protective agents • In vitro culture of OSCs and SSCs • Transplantation of cryopreserved cortical tissue pieces has given promising results. However, it is an invasive and expensive procedure. • Only option available is semen banking for men. Use of cryopreserved testicular tissue is not yet available in the clinics |