Stem cells and reproduction

Reproductive TractStem cells &Implication toReproductive Medicine臺 中 榮 民 總 醫 院 婦 產 部陳 明 哲 醫 師

Introduction• ESC, iPS, Adult SC• Ovarian Stem Cell and Transplantation• Uterine Stem Cell – Endometrium, Tube• Placental Stem Cell – Amnion, A Fluid• Fetal stem cell and F-M trafficking• Reproductive Cancer SC• Conclusion

Stem cellsDifferent SC types withdifferent characteristics withadvantages and disadvantages

• Undifferentiated cells1. Self-renewal: reproducing themselves2. Multi-lineage differentiation into manydifferent cell types3. In vivo functional reconstitution of a giventissue: producing at least one type ofhighly differentiated cells

• Embryonic stem cells1. Derived from the inner cell mass of theblastocysts2. Isolated from mouse in 1981, human in1998 -> developmental potential to formtrophoblast and derivatives of all threegerm layers in vitro.

• Research on embryonic stem cells raisesthe possibility of ‘designer’ tissue andorgan engineering.

• Even if those embryos are surplus torequirements for assisted reproductionand destined for destruction->Ethical considerations question theinstrumental use of embryos for theisolation of stem cells.

• One alternative is to explore the use ofadult stem cells; however, their fullpotential remains to be determined.

• Nearly all postnatal organs and tissuescontain populations of stem cells-> have the capacity for renewal afterdamage or ageing.• In the past several years, studies on adultstem cell plasticity revealed-> stem cells are able to (trans-)differentiate into other cell types in newlocations, in addition to their usual progenyin their organ of residence.

• Bone marrow derived stem cellBMCs (HSCs, MSCs) differentiate intoskeletal myoblasts, endothelium, cardiacmyoblasts, renal parenchymal, hepaticand biliary duct epithelium, lung, skin, GItracts, pancreas and CNS etc.--> BMD-SC may be involved in theregeneration of damaged tissue--> the possibility of repairing anindividual’s failing organ by BMT.

Stem cell niche

• The adult stem cells are responsible forthe growth, homeostasis and repair ofmany tissues.• How can they balance self-renewal withdifferentiation, and make the properlineage determination?

• In normal adult tissues, stem cells areultimately controlled by the integration ofintrinsic factors and extrinsic factors.(intrinsic: nuclear transcription factor)(extrinsic: cell-cell contact, growth factor,other external influence)

• Schofield (1978) proposed the stem cellniche hypothesis -> stem cells residewithin fixed compartments, or niches.1. This microenvironment, consisting ofspecialized cells, secretes signals andprovides cell surface molecules2. Control the rate of stem cell proliferation,determine the fate of stem cell progeny,and protect stem cells from death.

• Mammalian stem cells niches have beendescribed in the hematopoietic, neural,epidermal, and intestinal systems.• Recent work has revealed that theinteractions between stem cells and theirniches may be more dynamic thanoriginally believed.

• For example• Hematopoietic stem cells may occupytwo anatomically and physiologicallydistinct niches,-> an osteoblast niche and a vascularniche, and shuttle between them.• The vascular niche might explain stemcell survival in extramedullaryhaematopoietic sites, such as the liverand spleen.

A. Germline stem cells in thepostnatal ovary in mammal

• Germline stem cells (GSCs)1. The self-renewing population of germcells that serve as the source forgametogenesis.2. GSCs in Drosophila females maintainoocyte production in adult ovaries.3. Vertebrates? Mammals?

• A long-held dogma in ovarianbiology in mammals1. Females are born with a finite populationof nongrowing primordial follicles.2. Oocyte numbers decline throughoutpostnatal life, eventually leaving theovaries devoid of germ cells.3. In humans, the decline in oocytesnumbers is accompanied by exhaustionof the follicle pool and menopause beforethe end of life.

Joshua Johnson et al. providemurine evidence to challenge thisdoctrine (2004) _ MGH, Harvard

1. The numbers of healthy anddegenerating follicles in ovaries ofC57BL/6 mice were counted.2. The numbers of nonatretic quiescent andearly-growing prenatal follicles in singleovaries were higher than expected.3. The rate of depletion in the immatureovary was less than anticipated.

• The existence of proliferative GSCs thatgive rise to oocytes and follicleproduction in the postnatal period ofmammalian (murine) ovary.

• Bukovsky claimed to identifyGSCs and formation of newprimary follicles in adult humanovaries (2004)

1. Cytokeratin-positive mesenchymal cellsin ovarian tunica albuginea differentiateinto ovarian surface epithelium (OSE)cells by a mesenchymal–epithelialtransition.2. Germ cells can originate from surfaceepithelial cells which cover the tunicaalbuginea.

• The data also indicate that the pool ofprimary follicles in adult human ovariesmay not represent a static, but rather adynamic population of differentiating andregressing structures.

• These studies1. Existence of proliferative germ cells thatsustain oocyte and follicle production inthe postnatal mammalian ovary2. Oocytes are continuously formed in theadult.3. Function of donor-derived oocytesremains to be determined

A’. Origin of germ cellsin adult ovary

• The origin of oocytes in ovaries of adultmammalian females has been disputed forover 100 years.Set aside from embryonic cellIn vitro from ESCIn vitro from OSE• From BM / PB

• Weismann’s theory (In the 19thcentury)

1. Before embryonic cells becomecommitted along specific pathways, a setof germ cells is set aside, which aredestined to give rise to the gametes.2. This theory was not questioned until the1970s.

• In the early 2000s, evidence confirmedthat functional mouse oocytes and spermcan be derived from mouse embryonicstem cells in culture.

• Toyooka reported embryonic stem cellscan form germ cells (participate inspermatogenesis) in vitro.

• Geijsen found that injecting these culturedhaploid male gametes into unfertilized egg ledto embryo development to the early blastocyststage.

• Hubner reported that mouse embryonicstem cells in culture can develop intooogonia that enter meiosis and recruitadjacent cells to form follicle-likestructures and later developed intoblastocysts.

• Bukovsky proposed that in adult humanfemales, the OSE was a source of germ cells.(2004)

• In 2004, this group demonstrated in vivothat new primary follicles differentiatedfrom the adult human OSE, which arisesfrom cytokeratin-positive mesenchymalprogenitor cells residing in the ovariantunica albuginea.• In 2005, in-vitro culture of human OSEcells confirmed their in-vivo observationsthat the OSE is a bipotent source ofoocytes and granulosa cells.

• Johnson (2005) reported that mammalianoocytes originate from putative germ cellsin bone marrow and are distributedthrough peripheral blood to the ovaries.

• Bone marrow transplantation restoresoocyte production in wild-type micesterilized by chemotherapy, as well as inataxia telangiectasia-mutated genedeficientmice, which are otherwiseincapable of making oocytes.• Donor-derived oocytes are also observedin female mice following peripheral bloodtransplantation.

• It was suggested that bone marrow is apotential source of germ cells that couldsustain oocyte production in adulthood.

• The same group (2007) reported that bonemarrow transplantation generatesimmature oocytes and rescues long-termfertility in a preclinical mouse model ofchemotherapy-induced premature ovarianfailure.

• However, these studies are challenged bysome.• Wagers’ team established transplantationand parabiotic mouse models to assessthe capacity of circulating bone marrowcells to generate ovulated oocytes, both inthe steady state and after induced damage.

1. Their studies showed no evidence thatbone marrow cells, or any other normallycirculating cells, contribute to theformation of mature, ovulated oocytes.2. Instead, cells that travelled to the ovarythrough the bloodstream exhibitedproperties characteristic of committedblood leukocytes.

B. Ovarian tissuetransplantation

• Ovarian transplantation has a long history,traced back 200 years. However, therewas little progress until the middle of the20th century.

• Oktay and Karlikaya have reported thatovulation occurred after laparoscopictransplantation of frozen-thawed ovariantissue to the pelvic side wall in a 29-yearoldpatient who had undergone salpingooophorectomy.(NEJM, 2000)

• In 2004, the same group reported anothercase in which a four-cell embryo wasobtained from 20 oocytes retrieved fromtissue transplanted beneath the skin in apatient who had chemotherapy-inducedmenopause.

• The same year, a live birth after ovariantissue transplant was reported in anonhuman primate.

• In 2004, a successful pregnancy and livebirth after orthotopic transplantation ofcryopreserved ovarian tissue was reportedby Donnez.• In that case, a patient whose ovaries weredamaged by cancer chemotherapyreceived frozen-thawed ovarian tissuetransplantation.

• These findings give new hope for fertilitypreservation, including immature oocyteretrieval, in-vitro maturation of oocytes,oocyte vitrification or embryocryopreservation.

• However, one major concern overorthotopic auto-transplantation is thepotential risk that the frozen-thawedovarian cortex might harbor malignantcells.• There is the potential that such cells couldinduce a recurrence of disease after reimplantation.

• Silber (2005) reported that a 24-year-oldwoman gave birth after a transplant ofovarian cortical tissue from hermonozygotic twin sister.• Donnez (2007) reported another case ofsuccessful allograft of ovarian cortexbetween two genetically nonidenticalsisters.

• Silber (2007) reported 10 more successfulovary transplants in monozygotic twins afterpremature ovarian failure in one twin; twohealthy babies have been delivered, andanother three pregnancies are ongoing.

• Ovarian tissue transplantation not onlybrings hope to cancer patients, but also tothose with ovarian dysgenesis orpremature ovarian failure.

C. Stem cells in the uterus

• The uterine endometrium inmammals1. One of the most dynamic human tissue.2. Consists of a glandular epithelium andstroma that are completely renewed ineach monthly menstrual cycle.

• Endometrial stem cells1. Thought to reside in the basalis layer2. Serve as a source of cells thatdifferentiate to form the new endometrium

• Under systemic hormonal changes, suchas the cyclic increase in the serum level ofestradiol, stem cells migrate.• Give rise to a group of progenitor cells thatbecome committed to specific types ofdifferentiated cells, for example epithelial,stromal and vascular, within a certainmicroenvironment.

• These endogenous stem cells allow therapid regeneration of the endometriumnecessary to support pregnancy.

• Two studies from different labsprovided evidence for the origin ofthis cyclic renewal (2004)Yale – TaylorMonash - Gargett

• A team led by Gargett demonstrated (2004)that human endometrium contains smallpopulations of epithelial and stromal stemcells responsible for cyclical regenerationof endometrial glands and stroma and thatthese cells exhibited clonogenicity.

• In 2006, Gargett’s team used labelretainingcell (LRC) approach to identifysomatic stem/progenitor cells and theirlocation.• The presence of both epithelial andstromal LRC in mouse endometrium,which suggests that these stem-like cellsmay be responsible for endometrialregeneration.

• In 2008, Dimitrov et al also demonstratedthat the human endometrium contains alow number of cells with the characteristicsof endometrial stromal stem/ progenitorcells, which seem to belong to the familyof the mesenchymal stem cells (MSCs).

• In 2004 Taylor et al found that bonemarrow is an exogenous source ofendometrial cells.• They provided evidence of endometrialregeneration in bone marrow transplantrecipients who received marrow from asingle-HLA antigen mismatched donorBMT for leukemia.

• Donor-derived endometrial epithelial cellsand stromal cells were detected inendometrial samples of bone marrowrecipients by RT-PCR andimmunohistochemistry.• These cells appeared histologically to beendometrial epithelial and stromal cellsand also express appropriate markers ofendometrial cell differentiation.

• Taylor (2007) reported that in murinemodel after BMT, male donor-derivedbone marrow cells were found in theuterine endometrium of female mice, and,although uncommonly (

• Taylor (2007) et al also generatedexperimental endometriosis in a mousemodel by ectopic endometrial implantationin the peritoneal cavity and detected LacZexpressing cells in the wild-type ectopicendometrium after BMT from LacZtransgenic mice.• The result showed that bone marrowderivedcells also contribute endometriosis.

• In 2009, a group from Japan also reported thatbone marrow-derived cells from human maledonors can compose endometrial glands infemale transplant recipients.

• It was suggested that1. The repopulation of endometrium withbone marrow-derived stem cells may beimportant to normal endometrialphysiology2. May help to explain the cellular basis forthe high long-term failure of conservativealternatives to hysterectomy.

• The endometrium may regenerate afterresection or ablation from a stem cellsource outside of the uterus.• These findings have potential implicationsfor the treatment of uterine disorders.• These data support a new theory for thecause of endometriosis, which may haveits origin in ectopic trans-differentiation ofstem cells.

• In 2007 two studies determined theexistence of a small population ofmultipotent stem cells in endometriumYale – TaylorMonash - Gargett

• The Gargett’s lab collected humanendometrial tissue from reproductive-agedwomen, and prepared human endometrialstromal cell cultures.• Endometrial stromal cells were incubatedwith adipogenic, osteogenic and myogenicdifferentiation induction media for 4 weeks-> a subset of endometrial stromal cellsdifferentiate into cells of adipogenic,osteogenic, myogenic and chondrogeniccell lineages.

• Taylor’s lab also collected endometrialtissue from reproductive-aged women andmonolayer endometrial stromal cell (ESC),myometrial, fibroid, fallopian tube, anduterosacral ligament tissue cultures weregenerated.

• These cells were cultured in a definedchondrogenic media containingdexamethasone and transforming growthfactor for 21 days->analyzed for markers of human articularcartilage, including sulfatedglycosaminoglycans and type II collagen.

• Cultured endometrial derived stem cells(EDSCs) contain cells that can bedifferentiated into chondrocytes.

• Taylor group has recently reported thatEDSCs can be differentiated into neuronswhich produce dopamione and have thepotential to treat Parkinson’s disease.

Thus1. Since endometrium can easily beobtained, it may represent a newpotential source of pluripotent cells.2. Endometrial biopsy could become animportant source of stem cells for futurecell-based therapies.

D. Placenta and stem cells

Placenta and Hematopoiesis• Over the last 30 years, colonization hasbeen a long accepted theory-> the yolk sac was the sole source ofhematopoiesis in the mammalian embryo.

• The embryonic yolk sac-derived HSCscolonized fetal liver to initiate definitivehematopoiesis• Subsequently colonize bone marrow at theneonatal stages to support adulthematopoiesis.

• In the 1990s, accumulating evidencelocated hematopoiesis to another site inthe aorta-gonad-mesonephros (AGM) ofmouse embryos.

• A 2003 study indicated that1. The placenta contains a high frequencyof multipotential clonogenic progenitorsincluding CFU-GMs, CFU-GEMMs, BFU-Es and HPP-CFCs.2. The study results suggestthat the placenta mayfunction as a hematopoieticorgan during development.

• In 2005, two studies simultaneouslyreported that HSCs activity can bedetected in the mid-gestation placentallabyrinth region.• The onset of HSC activity in the placentacoincides with that in the AGM region andthe yolk sac.

• The expansion of the HSC pool in theplacenta occurs prior to and during theinitial expansion of HSCs in the fetal liver.• The size of the placental HSC pooldiminished, whereas the HSC pool in thefetal liver continues to expand.

• in 2004, some groups identified andisolated cells with MSC-like potency inhuman placenta.

• In recent years, the Huang group reportedthat placenta-derived multipotent cells candifferentiate into hepatocyte-like cells,neuronal and glial cells when the cellscultured under appropriate conditions invitro.• The placenta may be another source ofmultipotent stem cells.

E. Stem cell transfer fromthe fetus

• The presence of fetal cells in maternalcirculation has now been confirmed bymany investigators.

• Microchimeric cells of fetal origin havebeen identified in the peripheral blood ofpatients with the autoimmune diseasesystemic sclerosis (SSc).• However, it has not been determined ifthese cells are integrally involved in thepathogenesis of SSc, or if fetalmicrochimeric cells are just a marker ofinflammation.

• Increased numbers of microchimeric fetalcells have been identified in somediseases of pregnancy, for examplepreterm labor, preeclampsia andaneuploidy.

• However, there is speculation that theincreased number of fetal microchimericcells in the maternal circulation is areflection of the abnormalities within thestructure of the placenta, and not directlyrelated to the disease process.

• Bianchi (2001) discovered thatmale cells were seen in thyroidsections in women, presumablyfrom their sons.

• They reported that male cells were seenindividually or in clusters in all thyroiddisease from which biopsies wereexamined.• They were not restricted to inflammatorythyroid diseases.

• In one patient with a progressivelyenlarging goiter, they noted fullydifferentiated male thyroid follicles closelyattached to and indistinguishable from therest of the thyroid.

• Later on, this team reported that XY+microchimeric cells in maternal tissue,acquired most likely through pregnancy,express leukocyte, hepatocyte andepithelial markers.• The study also showed that hepatocytes offetal stem cell origin were identified in livertissue of one woman with liver injury andanother woman following hepatictransplantation.

• The results suggest that pregnancy mayresult in the physiologic acquisition of afetal cell population with the capacity formultilineage differentiation.

• These findings suggest that in a state inwhich the tissue injury is chronic, fetal cellmicrochimerism may be established morefrequently, or more easily and alsosuggests that microchimeric cells areinvolved in tissue repair.

• In 2008, two groups reported interestingstudies describing the contribution of fetalstem cells to cancer.

• One group investigated microchimeric fetalcells clustered at sites of tissue injury inthe lung decades after known malepregnancy.• Male cells were identified in lung/thymustissue from all women with sons.

• The male cells in the lung were clusteredin tumors rather than in surroundinghealthy tissues.• These male presumed-fetal cells wereidentified in pathological postreproductivetissues, in which they were more likely tobe located in diseased tissues at severalfoldhigher frequency than normal tissues.

• It is suggested that fetal cells are presentat sites of tissue injury and stem cells maybe, either recruited from marrow or havingproliferated locally.

• Breast carcinomas associated withpregnancy display a high frequency ofinflammatory types, multifocal lesions andlymph node metastasis.• Another group from France questionedwhether fetal stem cells are involved inthis disease process.

• They analyzed women presenting withcarcinomas who were pregnant with malefetuses.• The results showed that the presence offetal cells in pregnancy- associated breastcarcinoma is a frequent phenomenon.• These cells were predominantly part of thetumor stroma and could contribute to thepoorer profile of these carcinomas.

F. Cancer stem cells inreproductive tract

• Cancer stem cells (CSCs)1. Rare cell population in cancer withindefinite potential for self-renewal2. They are proposed to be the cancerinitiatingcells responsible fortumorigenesis and contribute to cancerresistance.3. How CSCs form? – alteration of selfrenewalpathway?

• The best known and most comparablepairs of somatic and CSCs are HSCs andleukemic stem cells (LSCs).

• Recently CSCs have been positivelyidentified and successfully isolated from alarge number of cancers.

• Ovarian cancer is an extremely aggressivedisease.• The cellular mechanisms underlying theincreasing aggressiveness associated withovarian cancer progression are poorlyunderstood.

• Epithelial ovarian cancers (EOCs) havebeen thought to arise from the simpleepithelium lining the ovarian surface orinclusion cysts-> the major subtypes of EOCs showmorphologic features that resemble thoseof the mullerian duct-derived epithelia ofthe reproductive tract.

HOX genes

1. Normally regulate mullerian ductdifferentiation, are not expressed innormal OSE2. But expressed in different EOC subtypesaccording to the pattern of mullerian-likedifferentiation of these cancers.

• Ectopic expression of Hoxa 9 intumorigenic mouse OSE cells gave rise topapillary tumors resembling serous EOCs.• In contrast, Hoxa10 and Hoxa11 inducedmorphogenesis of endometrioid- like andmucinous-like EOCs, respectively.

• Hoxa7 showed no lineage specificity, butpromoted the abilities of Hoxa9, Hoxa10,and Hoxa11 to induce differentiation alongtheir respective pathways.

• Stem cell transformation may bethe underlying mechanism leadingto ovarian cancer (2005).

• The study showed that a singletumorigenic clone was isolated among amixed population of cells derived from theascites of a patient with advanced ovariancancer.• During the course of the study, anotherclone underwent spontaneoustransformation in culture, providing amodel of disease progression.

• The tumorigenic clones, even on serialtransplantation, continue to establishtumors, thereby confirming their identity astumor stem cells.

• These findings suggest1. Stem cell transformation can be theunderlying cause of ovarian cancer.2. Progenitor cell transformation define theincreasing aggression that ischaracteristically associated with thedisease.

Side Population• Many types of stem cells use a multidrugresistance (MDR) pump to rid themselvesof chemicals, including nuclear dyes.• This property facilitates fluorescenceactivated cell sorting of those rare cellscapable of nuclear dye exclusion, whichhave been termed side-population cells.• Side-population cells exhibit many stemcell-like properties

Conclusion

• We are just beginning to understand stemcells, and many key questions remain.• Stem cells may play an important role innormal uterine and ovarian physiology.• They likely are involved in the response ofthese tissues to injury and disease.

• Stem cells likely play a role in pathology ofthe reproductive tract. Stem cells give riseto cancers and endometriosis.• A better understanding of stem cell biologymay prove helpful in the treatment of theseconditions.

• Finally the fetus, placenta and even theendometrium are all sources of stem cells.• Endometrial-derived stem cells mayprovide an immunologically matchedsource of multipotent stem cells for tissueengineering and regenerative medicine.