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Stem Cells in Obstetrics and Gynaecology Answers to Multiple Choice Questions for Vol. 18, No. 6
Best Practice & Research Clinical Obstetrics and Gynaecology Vol. 19, No. 1, pp. A11–A21, 2005 doi:10.1016/j.bpobgyn.2005.02.001 available online at http://www.sciencedirect.com
Stem Cells in Obstetrics and Gynaecology Answers to Multiple Choice Questions for Vol. 18, No. 6 1. (a) F
(b) F
(c) F
(d) T
(e) F
Explanation: (“d” is true: Pluripotency or the ability to form several cell types of all 3 germ layers is not possible in umbilical cord cells because they are not of early embryonic origin) 2. (a) F
(b) F
(c) T
(d) F
(e) F
Explanation: (“c” is true: Teratomas are produced in SCID mice only from hESCs as they are pluripotent) 3. (a) F
(b) F
(c) F
(d) F
(e) T
Explanation: (Answer: e. Animal oocytes (rabbit and cattle) have been successfully used to reprogram human adult somatic nuclei, but this will still be considered as human therapeutic cloning and is fraught with many ethical issues related to cross-species fertilization or cell union). 4. (a) F
(b) T
(c) F
(d) F
Explanation: “b” is true: hESCs can be induced to enter a program of differentiation in vitro through the formation of embryoid bodies (EBs), in which they differentiate as tissue-like spheroids in suspension culture. Human EB formation can be acquired by culturing hESC aggregates in suspension. 1521-6934/$ - see front matter
A12 Appendix
5. (a) F
(b) F
(c) T
(d) F
Explanation: “c” is true: Unlike mouse ESCs, hESCs can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of hESCs to derive early human trophoblasts is particularly valuable, because they are difficult to obtain from other sources and are significantly different from mouse trophoblasts.
6. (a) F
(b) F
(c) T
(d) F
Explanation: “c” is true: In the early stages of embryonic development, vessel formation occurs by a process referred to as vasculogenesis, in which mesodermally-derived endothelial cell progenitors undergo de-novo differentiation, expand and coalesce to form a network of primitive tubules
7. (a) F
(b) F
(c) T
(d) T
(e) T
Explanation: Fetal stem cells can be isolated from fetal blood and bone marrow as well as other fetal tissues, including liver and kidney. In contrast, embryonic germ cells can be obtained from the gonadal tissues of post-implantation embryos. Stem cells obtained from fetal blood and tissues are believed to have similar properties and immunophenotype to comparable adult tissue-derived stem cells, while their development potential is more restricted than pluripotent embryonic stem cells. Fetal stem cells have advantages over their adult counterparts, including better engraftment, greater multipotentiality, higher proliferative potential and lower immunogenicity. Fetal blood and liver are accessible sources of fetal stem cells that might be collected for autologous use in ongoing pregnancies.
8. (a) F
(b) F
(c) F
(d) F
(e) T
Explanation: Fetal haemopoietic stem cells were originally defined by their biologic properties and by the expression of the “haemopoietic” CD34 antigen, as well as the absence of markers such as CD38 and HLA-DR. First trimester fetal blood contains a higher frequency of CD34C cells than term gestation blood. The number of haemopoietic progenitors circulating in fetal blood peaks in the second trimester. Functional studies of HSC suggest that CD34C cells have a higher cloning efficiency and generate more progenitors than CD34C cells from
Appendix A13
adult bone marrow. There are significant differences between HSC isolated from, for example, fetal liver and adult bone marrow. Fetal blood HSC proliferate extensively in vitro and produce all haemopoietic lineages: CFUGEMM, CFU-GM, CFU-MK, BFU-MK and BFU-e, although the predominant progenitors grown are CFU-GEMM and BFU-e.
9. (a) T
(b) T
(c) T
(d) F
(e) F
Explanations: No matter what their site of origin, fetal MSC do not express markers associated with haemopoietic or endothelial differentiation. MSC have been isolated from fetal blood and bone marrow, as well as from liver, lung, pancreas and kidney. The earliest time at which MSC have been isolated in fetal blood is 7 weeks gestation, and very few MSC are present in the circulation from 14 weeks. Fetal bloodderived MSC are readily expandable in vitro with population doublings every 30 hours, compared to at best 48–72 hours for their adult counterparts, and display no obvious change in phenotype after 20 passages. More recent work suggests fetal blood-derived can differentiate into oligodendrocytes, although whether they can integrate into the damaged central nervous system in vivo and functionally improve outcome has yet to be demonstrated.
10. (a) F
(b) F
(c) F
(d) F
(e) T
Explanation: (a-e) Embryonic and fetal cells from all three germ layers have long been identified in the amniotic fluid [2-5].
11. (a) T
(b) T
(c) F
(d) T
(e) T
Explanation: (a) The origin(s) of the MSCs found in the amniotic fluid still remains to be determined. (b) Animal data have shown that amniotic fluid-derived MSCs proliferated significantly faster in culture than immunocytochemically comparable cells derived from fetal or adult subcutaneous connective tissue (Figure 1) [30] (c) Mesenchymal amniocytes are very abundant in the amniotic fluid, from which they can be easily isolated. (d) In humans, the expansion potential of mesenchymal amniocytes exceeds that of bone marrow-derived MSCs[19, 31]. (e) The phenotype of human mesenchymal amniocytes expanded in culture is similar to that reported for MSCs derived from second trimester fetal tissue and adult bone marrow [19, 28, 34].
A14 Appendix
12. (a) T
(b) F
(c) T
(d) T
(e) T
Explanation: (a) (b)
(c) (d)
(e)
The origin(s) of the embryonic-like stem cells found in the amniotic fluid still remains to be determined. A uniform and universal differentiation potential remains to be verified in these cells. So far, they have been shown to differentiate into muscle, adipogenic, osteogenic, nephrogenic, neural, and endothelial cells, but not necessarily from a uniform population of undifferentiated cells[21]. These cells are very scarce, representing zero (i.e. they cannot always be isolated from the amniotic fluid) to less than 1% of the cells present in amniocentesis samples[5, 20, 21]. The markers used to identify amniotic embryonic-like stem cells can also be expressed, alone or in various combinations, in embryonic germ cells; embryonic fibroblasts; embryonal carcinoma cells; mesenchymal stem cells; hematopoietic stem cells; ectodermal, neural, and pancreatic progenitor cells; and fetal and adult nerve tissue; among others[5, 28, 36-38]. These cells seem to be clonogenic, as at least the Oct-4 positive ones express cyclin A, a cell cycle regulator[5, 40].
13. (a) F
(b) F
(c) T (d) F
(e) F
Explanation: (a). In a recent study published (36), the rate of neutrophil recovery for UCB versus bone marrow was either similar or slower when a constant number of cells were delivered. (b) Acute GVHD with UCB is lower by w50%. (c) One of the major advantages of UCB for BM transplants is that both acute and chronic GVHD are reduced. Related, UCB is more tolerant of mismatches at the HLA loci. (d) Despite the fact that GVHD is decreased, patient survival is lower over a 5-year period when compared to BM. (e) There are more stem cells per ml of blood collected from UCB versus BM, but UCB is limited to the amount found in a single cord and placenta.
14. (a) F
(b) F
(c) T
(d) F
(e) F
Explanation: (a) None of these answers can be absolutely eliminated. Transdifferentiation assumes the original genetic program is stripped and the cell is re-programmed. Although possible, as shown by cloning experiments, the frequency of non-blood cells arising from blood samples exceeds the expected frequency of transdifferentiation. (b) Although this has not been ruled out, labs have looked for ES-like cells based on markers (OCT-4, SSEA1, and SSEA4) and nothing definitive has resulted. (c) Fusion of donor cells with recipient cells for both mouse and human in a number of tissues has been clearly demonstrated. (d) For example, vimentin, a marker for mesenchymal cells also appears on endothelial cells. (e) Placing cells into alternative environments, whether on vivo or in vitro can alter the
Appendix A15
properties of the cell. This may be advantageous for producing a wider range of cell types. This mechanism remains unproven. 15. (a) T
(b) F
(c) F (d) F
(e) F
Explanation: (a) As discussed in this chapter, the presence of a definitive mesenchymal cell identical to the type found in bone marrow is debatable. The cell surface markers and the numbers of mesenchymal cells from UCB versus bone marrow differ. The one strong similarity is that both bone marrow and UCB contain a cell capable of differentiating into non-blood cell types. (b) The CD45 status of the UCB mesenchymal cell has not be defined. Different groups have isolated mesenchymallike cells that are either CD45C or CD45-. (c) Bone marrow contains a higher frequency of mesenchymal cells. (d) The mesenchymal-like cells identified in UCB grow at a much slower rate than bone marrow mesenchymal cells under the same tissue culture conditions. (e) Using the bone marrow mesenchymal cell as a strict model for all mesenchymal cells, the UCB mesenchymal cell does not hold up.
16. (a)F 17.
(a) F
18. (a)F
(b) F
(c) F
(b) F (b) F
(d) T
(c) T
(c)F
(e) F
(d)F
(d) T
(e) F (e) F
[NB: d is the INCORRECT statement, while the rest are correct - see question] 19. (a) F
(b) T
(c) F
(d) F
Explanation: Embryonic stem (ES) cells are pluripotent cells that derive from the ICM of a blastocyst-stage embryo. These cells remain undifferentiated during prolonged propagation in vitro and retain a stable normal karyotype. Human ES cells were derived from in vitro fertilized oocytes grown in culture to the blastocyst stage. These cells were shown to fulfil the essential criteria for classification as ES cells.
20. (a) T
(b) T
(c) T
(d) T
(e) T
f) F
Explanation: Human ES cells have been shown to differentiate spontaneously into all three embryonic germ layers in vivo and in vitro, and also to respond to inductive signals and initiate a specific developmental program accordingly.
A16 Appendix
21. (a) T
(b) F
(c) F
(d) F
Explanation: One of the X chromosomes is inactivated during early development of female mammals. X inactivation normally occurs randomly, with equal probabilities for the paternal- or the maternal-inherited X chromosome to undergo inactivation. Once it has been inactivated, the X chromosome remains inactive through subsequent cell divisions.
22. (a) T
(b) T
(c) F
(d) F
(e) T
Explanation: (a) Engraftment of allogeneic or even xenogeneic cells is facilitated by the phenomenon of immunologic tolerance. This allows engraftment without rejection of the donor cells and without the need for toxic immunosuppression. (b) The most favorable circumstance for therapeutic levels of donor cell engraftment are in disorders (or genetically impaired animal models) in which there is a selective survival or proliferative advantage favoring donor cells. Examples include human and mouse Severe Combined Immunodeficiency Syndrome (SCID) and murine c-kit deficiency. (c) There is little to support the presence of significant space in the fetal microenvironment. “Space” is a function of stromal formation with the development of “niches” for stem cell occupation, the number of host stem cells available to occupy the niches, and the dissociation coefficient for donor versus host stem cells once a niche is occupied. The fetus appears to have an excess of highly competitive host stem cells, providing one of the primary challenges to successful stem cell engraftment. (d) The immunologic and developmental “window of opportunity” is prior to the maturation of the host immune system and during the period of stem cell migration and engraftment of tissue compartments. Thus, early gestational transplantation is optimal. (e) The small size of the fetus provides the opportunity to transplant much higher doses of cells on a relative scale favoring competitive engraftment.
23. (a) F
(b) F
(c) F
(d) T
(e) T
Explanation: (a) In utero stem cell transplantation should no longer be performed clinically based on inadequate rationale and without relevant and supportive experimental data. (b) There are currently no data in a large animal model with competitive hematopoiesis demonstrating adequate engraftment to support success in humans with haemoglobinopathy. There has been a number of reports of failure in this category of disease. Until further supportive experimental data are
Appendix A17
generated, the haemoglobinopathies are not an appropriate target for this approach. (c) As in (b) above. In addition, no studies have convincingly demonstrated the ability of transplanted cells to cross the blood brain barrier in the fetus. Thus lysosomal storage disorders with neurological manifestations represent one of the least attractive disease categories for this approach. (d) The only disorders in which there is adequate experimental and clinical support for current treatment by in utero stem cell transplantation is SCID (nonadenosine deaminase deficiency types). There are a few other disorders in which the same rationale may apply but further experimental work is needed. (e) This is the only circumstance in which the in utero stem cell transplantation should be clinically applied in the future. Until we are able to achieve therapeutic levels of engraftment in large animal models (other than the sheep) with relevant barriers to engraftment, experimental work in humans should not be performed.
24. (a) F
(b) F
(c) T
(d) F
(e) T
Explanation: (a)
(b)
(c) (d)
(e)
False. While it is true that fetal cells do circulate from prior pregnancies for decades, these cells are presumably PAPCs or leukocytes. Strategies can be developed to isolate only short-lived fetal cells, such as nucleated erythrocytes or trophoblasts that represent the current pregnancy. False. Even if fetal cells isolated from maternal blood do not divide in culture, one could still perform prenatal genetic diagnosis using fluorescent in situ hybridization interphase analysis of fetal nuclei. Using this method, one can count the number of chromosome-specific signals and recognize the common aneuploidies. True. The low number of fetal cells present in most maternal blood samples is the most significant technical limitation to applying this method clinically. False. Although it is somewhat easier to analyze the genetic content of isolated fetal cells by PCR than FISH, genetic analysis has never been the limiting factor. The literature contains many reports on the genetic analysis of fetal cells isolated from maternal blood. True. However, investigators have gotten around this problem by selecting antigens that are expressed in most fetal cells as well as a subset of maternal cells. This results in an isolated population of cells that has low fetal cell purity but still recovers many fetal cells.
25 (a) T
(b) F
(c) T
(d) F
(e) F
Explanation: (a)
True. Thyroid diseases are more common in women than men, and fetal cell microchimerism is increased in thyroid specimens taken from women with both autoimmune and non-autoimmune thyroid conditions.
A18 Appendix
(b) False. Although breast cancer is a condition that occurs almost exclusively in women, to date there have been no studies performed to determine whether fetal cells can be detected in breast tissue from normal or pathological specimens. (c) True. This condition has been extensively studied and multiple groups have shown an increased number of fetal cells in affected tissues compared with controls of similar parity. (d) False. Surprisingly, even though primary biliary cirrhosis almost exclusively occurs in middle-aged women, there does not appear to be an increased number of fetal cells present in the livers of affected women. It is of interest, however, that the liver seems to be an organ in which fetal cells are detectable in appreciable numbers in all parous women. (e) As of the writing of this manuscript, false, in that there are no data available. Pulmonary hypertension is also more common in women than men. This is a good candidate disease to study further.
26. (a) F (a)
(b)
(c)
(d) (e)
(b) T
(c) T
(d) F
(e) F
Explanation: False. Fetal cell microchimerism results equally from male and female fetuses. However, it is easier to demonstrate that fetal cells are present in maternal tissue when the fetus is male by documenting evidence of a Y chromosome, which the mother lacks. Male fetuses are therefore used to prove that microchimerism exists. True. A meta-analysis has shown that women with a history of having had a fetal loss were more likely to have evidence of fetal cell microchimerism than otherwise matched controls. This study did not distinguish between fetal loss due to abortion and fetal loss due to miscarriage. True. See above. It is tempting to speculate that microchimerism is more likely to result from an abortion in which there is a non-physiologic breach of the feto-maternal barrier at a time when the fetal blood cells are still circulating and functional. A miscarriage is preceded by fetal death, and presumably, death of the fetal blood cells. Thus, the answer to c may be “false” but this needs further study. False. Although not specifically discussed in the manuscript, fetal cell microchimerism also occurs in the rhesus monkey. False. Although there is evidence to suggest that HLA class II compatibility between a woman, her fetus, and her own mother plays a role in the development and maintenance of microchimerism, it is not required for the development of microchimerism.
27. (a) F
(b) T
(c) T
(d) F
(e) F
Explanation: (a) NSC have been identified throughout the early fetal brain. However in the adult mature brain NSC have been identified only in the subventricular zone
Appendix A19
(b)
(c)
(d) (e)
and the hippocampus. These areas support ongoing proliferation of neural stem cells that contribute to the generation of new neurones particularly following injury. Cells of neural morphology and phenotype have been derived from bone marrow and blood of human and rodents both in vitro and in vivo. This work is controversial, and several investigators have demonstrated that the apparent differentiation of bone-marrow to neural cells in vivo is actually caused by the fusion of a donor cell to a brain cell. NSC are highly proliferative and are commonly expanded as aggregates (neurospheres). Recently culture conditions have been established to grow them as monolayers. Importantly neurospheres do not just contain stem cells, but a population of stem cells with more differentiated progeny. NSC can generate the 3 major cell types of the CNS- neurones, oligodendrocytes and astrocytes. The other brain cell type, microglia, are derived from the blood. Although there are a variety of surface markers that enrich for NSC (eg Nestin, Musashi, Notch 1, CD133, etc) none is absolutely specific. The identification of NSC still relies on their ability to self renew and differentiate into neurones, oligodendrocytes and astrocytes.
28. (a) F
(b) F
(c) F
(d) T
(e) F
Explanation: (a) PVL is now infrequent (!2%) in extremely preterm infants. It is associated with a poor neurological prognosis. The commonest brain abnormality seen in preterm infants is diffuse increased signal intensity in the white matter on T2 weighted MR imaging. Although the pathological basis for this MR imaging finding is not confirmed. It is likely that it results from oligodendrocyte loss or injury. (b) Although HIE is a clear cause of cerebral palsy in some infants, the majority of infants do not have a clear birth insult although many have antenatal risk factors. (c) HIE typically leads to widespread cell loss with grey and white matter injury. Potential cell replacement strategies would need to consider this. (d) There are no neuroprotective treatments in widespread use for HIE at present. There are encouraging trials underway investigating the effect of brain cooling in such infants and the results awaited. In vivo studies suggest cooling reduces apoptotic cell death. (e) Most metabolic brain diseases are sporadic or autosomal recessive and so generally occur in previously unaffected families. If there is an affected sibling genetic/metabolic diagnosis may be possible antenatally. Many metabolic disorders have normal brain morphology and structure and so would not be diagnosed either with ultrasound or fetal MR. The majority of infants with metabolic brain diseases will present after the onset of symptoms.
A20 Appendix
29. (a) T
(b) F
(c) T
(d) T
(e) T
Explanation: (a)
(b)
(c)
(d)
(e)
The major advantage of perinatal cell therapy in metabolic disease is to intervene before considerable brain damage occurs. However the major challenge is to obtain an early accurate diagnosis. In addition NSC, unless genetically altered, may be vulnerable to the disease process itself, and even if successful would not alter systemic manifestations. There have been considerable advances in the genetic basis of metabolic diseases which aids diagnosis. However there remains a lack of knowledge of the cell and molecular interaction within the brain and the effects of the diseased brain environment. This knowledge would be necessary for the successful development of stem cell strategies. One would imagine the immature brain has more inherent plasticity and potential for repair, and it may well be that the environment of developing brain would contain the necessary cues for appropriate differentiation. However there is little systematic research studying the best developmental age for transplantation survival and integration. The immune system in fetal life and the early neonatal period is relatively naı¨ve. Again research is needed to support the assumption that grafting stem cells earlier may lead to improved graft tolerance and survival Undoubtedly the fetal or newborn brain is more accessible to direct injection of stem cells than the mature brain, but data suggests NSC are highly migratory and may actually home to sites of injury with simple injection into the CSF space. Also, as with gene therapy approaches there may be mechanical advantages to starting treatment in a smaller, rather than mature, brain in terms of the numbers of cells needed to populate a given area and instruct repair.
30. (a) T
(b) T
(c) F
(d) T
Explanation: (a) Commercially banked units are only for the use of the direct family and are unlikely to be used because the indications for BMT are rare (b) The commonest use for cord blood transplant is for acute leukaemia of childhood. This is a rare disease and is commonly cured by chemotherapy and transplant is rarely required (c) There are concerns that the cord blood of an infant who subsequently develops childhood leukaemia may contain markers of leukaemia predisposition. (d) Bacterial contamination with pathogens or low volume collections or clots in the collection are commonly found, especially if the person undertaking the collection is inexperienced.
Appendix A21
31. (a) T
(b) T
(c) F
(d) T
Explanation: (a) The midwife’s and obstetrician’s primary task is care of the infant/maternal pair (b) The interests of the cord collection may not best serve the interests of the donor infant (c) Bacterial contamination rates are a high as 30% in inexperienced hands but are reduced to less than 1% by dedicated trained staff, as demonstrated by the London Cord Blood Bank (d) The collection may be bacterially contaminated, of insufficient volume or contain large clots.
Stem Cells in Obstetrics and Gynaecology Answers to Multiple Choice Questions for Vol. 18, No. 6 1. (a) F
(b) F
(c) F
(d) T
(e) F
Explanation: (“d” is true: Pluripotency or the ability to form several cell types of all 3 germ layers is not possible in umbilical cord cells because they are not of early embryonic origin) 2. (a) F
(b) F
(c) T
(d) F
(e) F
Explanation: (“c” is true: Teratomas are produced in SCID mice only from hESCs as they are pluripotent) 3. (a) F
(b) F
(c) F
(d) F
(e) T
Explanation: (Answer: e. Animal oocytes (rabbit and cattle) have been successfully used to reprogram human adult somatic nuclei, but this will still be considered as human therapeutic cloning and is fraught with many ethical issues related to cross-species fertilization or cell union). 4. (a) F
(b) T
(c) F
(d) F
Explanation: “b” is true: hESCs can be induced to enter a program of differentiation in vitro through the formation of embryoid bodies (EBs), in which they differentiate as tissue-like spheroids in suspension culture. Human EB formation can be acquired by culturing hESC aggregates in suspension. 1521-6934/$ - see front matter
A12 Appendix
5. (a) F
(b) F
(c) T
(d) F
Explanation: “c” is true: Unlike mouse ESCs, hESCs can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of hESCs to derive early human trophoblasts is particularly valuable, because they are difficult to obtain from other sources and are significantly different from mouse trophoblasts.
6. (a) F
(b) F
(c) T
(d) F
Explanation: “c” is true: In the early stages of embryonic development, vessel formation occurs by a process referred to as vasculogenesis, in which mesodermally-derived endothelial cell progenitors undergo de-novo differentiation, expand and coalesce to form a network of primitive tubules
7. (a) F
(b) F
(c) T
(d) T
(e) T
Explanation: Fetal stem cells can be isolated from fetal blood and bone marrow as well as other fetal tissues, including liver and kidney. In contrast, embryonic germ cells can be obtained from the gonadal tissues of post-implantation embryos. Stem cells obtained from fetal blood and tissues are believed to have similar properties and immunophenotype to comparable adult tissue-derived stem cells, while their development potential is more restricted than pluripotent embryonic stem cells. Fetal stem cells have advantages over their adult counterparts, including better engraftment, greater multipotentiality, higher proliferative potential and lower immunogenicity. Fetal blood and liver are accessible sources of fetal stem cells that might be collected for autologous use in ongoing pregnancies.
8. (a) F
(b) F
(c) F
(d) F
(e) T
Explanation: Fetal haemopoietic stem cells were originally defined by their biologic properties and by the expression of the “haemopoietic” CD34 antigen, as well as the absence of markers such as CD38 and HLA-DR. First trimester fetal blood contains a higher frequency of CD34C cells than term gestation blood. The number of haemopoietic progenitors circulating in fetal blood peaks in the second trimester. Functional studies of HSC suggest that CD34C cells have a higher cloning efficiency and generate more progenitors than CD34C cells from
Appendix A13
adult bone marrow. There are significant differences between HSC isolated from, for example, fetal liver and adult bone marrow. Fetal blood HSC proliferate extensively in vitro and produce all haemopoietic lineages: CFUGEMM, CFU-GM, CFU-MK, BFU-MK and BFU-e, although the predominant progenitors grown are CFU-GEMM and BFU-e.
9. (a) T
(b) T
(c) T
(d) F
(e) F
Explanations: No matter what their site of origin, fetal MSC do not express markers associated with haemopoietic or endothelial differentiation. MSC have been isolated from fetal blood and bone marrow, as well as from liver, lung, pancreas and kidney. The earliest time at which MSC have been isolated in fetal blood is 7 weeks gestation, and very few MSC are present in the circulation from 14 weeks. Fetal bloodderived MSC are readily expandable in vitro with population doublings every 30 hours, compared to at best 48–72 hours for their adult counterparts, and display no obvious change in phenotype after 20 passages. More recent work suggests fetal blood-derived can differentiate into oligodendrocytes, although whether they can integrate into the damaged central nervous system in vivo and functionally improve outcome has yet to be demonstrated.
10. (a) F
(b) F
(c) F
(d) F
(e) T
Explanation: (a-e) Embryonic and fetal cells from all three germ layers have long been identified in the amniotic fluid [2-5].
11. (a) T
(b) T
(c) F
(d) T
(e) T
Explanation: (a) The origin(s) of the MSCs found in the amniotic fluid still remains to be determined. (b) Animal data have shown that amniotic fluid-derived MSCs proliferated significantly faster in culture than immunocytochemically comparable cells derived from fetal or adult subcutaneous connective tissue (Figure 1) [30] (c) Mesenchymal amniocytes are very abundant in the amniotic fluid, from which they can be easily isolated. (d) In humans, the expansion potential of mesenchymal amniocytes exceeds that of bone marrow-derived MSCs[19, 31]. (e) The phenotype of human mesenchymal amniocytes expanded in culture is similar to that reported for MSCs derived from second trimester fetal tissue and adult bone marrow [19, 28, 34].
A14 Appendix
12. (a) T
(b) F
(c) T
(d) T
(e) T
Explanation: (a) (b)
(c) (d)
(e)
The origin(s) of the embryonic-like stem cells found in the amniotic fluid still remains to be determined. A uniform and universal differentiation potential remains to be verified in these cells. So far, they have been shown to differentiate into muscle, adipogenic, osteogenic, nephrogenic, neural, and endothelial cells, but not necessarily from a uniform population of undifferentiated cells[21]. These cells are very scarce, representing zero (i.e. they cannot always be isolated from the amniotic fluid) to less than 1% of the cells present in amniocentesis samples[5, 20, 21]. The markers used to identify amniotic embryonic-like stem cells can also be expressed, alone or in various combinations, in embryonic germ cells; embryonic fibroblasts; embryonal carcinoma cells; mesenchymal stem cells; hematopoietic stem cells; ectodermal, neural, and pancreatic progenitor cells; and fetal and adult nerve tissue; among others[5, 28, 36-38]. These cells seem to be clonogenic, as at least the Oct-4 positive ones express cyclin A, a cell cycle regulator[5, 40].
13. (a) F
(b) F
(c) T (d) F
(e) F
Explanation: (a). In a recent study published (36), the rate of neutrophil recovery for UCB versus bone marrow was either similar or slower when a constant number of cells were delivered. (b) Acute GVHD with UCB is lower by w50%. (c) One of the major advantages of UCB for BM transplants is that both acute and chronic GVHD are reduced. Related, UCB is more tolerant of mismatches at the HLA loci. (d) Despite the fact that GVHD is decreased, patient survival is lower over a 5-year period when compared to BM. (e) There are more stem cells per ml of blood collected from UCB versus BM, but UCB is limited to the amount found in a single cord and placenta.
14. (a) F
(b) F
(c) T
(d) F
(e) F
Explanation: (a) None of these answers can be absolutely eliminated. Transdifferentiation assumes the original genetic program is stripped and the cell is re-programmed. Although possible, as shown by cloning experiments, the frequency of non-blood cells arising from blood samples exceeds the expected frequency of transdifferentiation. (b) Although this has not been ruled out, labs have looked for ES-like cells based on markers (OCT-4, SSEA1, and SSEA4) and nothing definitive has resulted. (c) Fusion of donor cells with recipient cells for both mouse and human in a number of tissues has been clearly demonstrated. (d) For example, vimentin, a marker for mesenchymal cells also appears on endothelial cells. (e) Placing cells into alternative environments, whether on vivo or in vitro can alter the
Appendix A15
properties of the cell. This may be advantageous for producing a wider range of cell types. This mechanism remains unproven. 15. (a) T
(b) F
(c) F (d) F
(e) F
Explanation: (a) As discussed in this chapter, the presence of a definitive mesenchymal cell identical to the type found in bone marrow is debatable. The cell surface markers and the numbers of mesenchymal cells from UCB versus bone marrow differ. The one strong similarity is that both bone marrow and UCB contain a cell capable of differentiating into non-blood cell types. (b) The CD45 status of the UCB mesenchymal cell has not be defined. Different groups have isolated mesenchymallike cells that are either CD45C or CD45-. (c) Bone marrow contains a higher frequency of mesenchymal cells. (d) The mesenchymal-like cells identified in UCB grow at a much slower rate than bone marrow mesenchymal cells under the same tissue culture conditions. (e) Using the bone marrow mesenchymal cell as a strict model for all mesenchymal cells, the UCB mesenchymal cell does not hold up.
16. (a)F 17.
(a) F
18. (a)F
(b) F
(c) F
(b) F (b) F
(d) T
(c) T
(c)F
(e) F
(d)F
(d) T
(e) F (e) F
[NB: d is the INCORRECT statement, while the rest are correct - see question] 19. (a) F
(b) T
(c) F
(d) F
Explanation: Embryonic stem (ES) cells are pluripotent cells that derive from the ICM of a blastocyst-stage embryo. These cells remain undifferentiated during prolonged propagation in vitro and retain a stable normal karyotype. Human ES cells were derived from in vitro fertilized oocytes grown in culture to the blastocyst stage. These cells were shown to fulfil the essential criteria for classification as ES cells.
20. (a) T
(b) T
(c) T
(d) T
(e) T
f) F
Explanation: Human ES cells have been shown to differentiate spontaneously into all three embryonic germ layers in vivo and in vitro, and also to respond to inductive signals and initiate a specific developmental program accordingly.
A16 Appendix
21. (a) T
(b) F
(c) F
(d) F
Explanation: One of the X chromosomes is inactivated during early development of female mammals. X inactivation normally occurs randomly, with equal probabilities for the paternal- or the maternal-inherited X chromosome to undergo inactivation. Once it has been inactivated, the X chromosome remains inactive through subsequent cell divisions.
22. (a) T
(b) T
(c) F
(d) F
(e) T
Explanation: (a) Engraftment of allogeneic or even xenogeneic cells is facilitated by the phenomenon of immunologic tolerance. This allows engraftment without rejection of the donor cells and without the need for toxic immunosuppression. (b) The most favorable circumstance for therapeutic levels of donor cell engraftment are in disorders (or genetically impaired animal models) in which there is a selective survival or proliferative advantage favoring donor cells. Examples include human and mouse Severe Combined Immunodeficiency Syndrome (SCID) and murine c-kit deficiency. (c) There is little to support the presence of significant space in the fetal microenvironment. “Space” is a function of stromal formation with the development of “niches” for stem cell occupation, the number of host stem cells available to occupy the niches, and the dissociation coefficient for donor versus host stem cells once a niche is occupied. The fetus appears to have an excess of highly competitive host stem cells, providing one of the primary challenges to successful stem cell engraftment. (d) The immunologic and developmental “window of opportunity” is prior to the maturation of the host immune system and during the period of stem cell migration and engraftment of tissue compartments. Thus, early gestational transplantation is optimal. (e) The small size of the fetus provides the opportunity to transplant much higher doses of cells on a relative scale favoring competitive engraftment.
23. (a) F
(b) F
(c) F
(d) T
(e) T
Explanation: (a) In utero stem cell transplantation should no longer be performed clinically based on inadequate rationale and without relevant and supportive experimental data. (b) There are currently no data in a large animal model with competitive hematopoiesis demonstrating adequate engraftment to support success in humans with haemoglobinopathy. There has been a number of reports of failure in this category of disease. Until further supportive experimental data are
Appendix A17
generated, the haemoglobinopathies are not an appropriate target for this approach. (c) As in (b) above. In addition, no studies have convincingly demonstrated the ability of transplanted cells to cross the blood brain barrier in the fetus. Thus lysosomal storage disorders with neurological manifestations represent one of the least attractive disease categories for this approach. (d) The only disorders in which there is adequate experimental and clinical support for current treatment by in utero stem cell transplantation is SCID (nonadenosine deaminase deficiency types). There are a few other disorders in which the same rationale may apply but further experimental work is needed. (e) This is the only circumstance in which the in utero stem cell transplantation should be clinically applied in the future. Until we are able to achieve therapeutic levels of engraftment in large animal models (other than the sheep) with relevant barriers to engraftment, experimental work in humans should not be performed.
24. (a) F
(b) F
(c) T
(d) F
(e) T
Explanation: (a)
(b)
(c) (d)
(e)
False. While it is true that fetal cells do circulate from prior pregnancies for decades, these cells are presumably PAPCs or leukocytes. Strategies can be developed to isolate only short-lived fetal cells, such as nucleated erythrocytes or trophoblasts that represent the current pregnancy. False. Even if fetal cells isolated from maternal blood do not divide in culture, one could still perform prenatal genetic diagnosis using fluorescent in situ hybridization interphase analysis of fetal nuclei. Using this method, one can count the number of chromosome-specific signals and recognize the common aneuploidies. True. The low number of fetal cells present in most maternal blood samples is the most significant technical limitation to applying this method clinically. False. Although it is somewhat easier to analyze the genetic content of isolated fetal cells by PCR than FISH, genetic analysis has never been the limiting factor. The literature contains many reports on the genetic analysis of fetal cells isolated from maternal blood. True. However, investigators have gotten around this problem by selecting antigens that are expressed in most fetal cells as well as a subset of maternal cells. This results in an isolated population of cells that has low fetal cell purity but still recovers many fetal cells.
25 (a) T
(b) F
(c) T
(d) F
(e) F
Explanation: (a)
True. Thyroid diseases are more common in women than men, and fetal cell microchimerism is increased in thyroid specimens taken from women with both autoimmune and non-autoimmune thyroid conditions.
A18 Appendix
(b) False. Although breast cancer is a condition that occurs almost exclusively in women, to date there have been no studies performed to determine whether fetal cells can be detected in breast tissue from normal or pathological specimens. (c) True. This condition has been extensively studied and multiple groups have shown an increased number of fetal cells in affected tissues compared with controls of similar parity. (d) False. Surprisingly, even though primary biliary cirrhosis almost exclusively occurs in middle-aged women, there does not appear to be an increased number of fetal cells present in the livers of affected women. It is of interest, however, that the liver seems to be an organ in which fetal cells are detectable in appreciable numbers in all parous women. (e) As of the writing of this manuscript, false, in that there are no data available. Pulmonary hypertension is also more common in women than men. This is a good candidate disease to study further.
26. (a) F (a)
(b)
(c)
(d) (e)
(b) T
(c) T
(d) F
(e) F
Explanation: False. Fetal cell microchimerism results equally from male and female fetuses. However, it is easier to demonstrate that fetal cells are present in maternal tissue when the fetus is male by documenting evidence of a Y chromosome, which the mother lacks. Male fetuses are therefore used to prove that microchimerism exists. True. A meta-analysis has shown that women with a history of having had a fetal loss were more likely to have evidence of fetal cell microchimerism than otherwise matched controls. This study did not distinguish between fetal loss due to abortion and fetal loss due to miscarriage. True. See above. It is tempting to speculate that microchimerism is more likely to result from an abortion in which there is a non-physiologic breach of the feto-maternal barrier at a time when the fetal blood cells are still circulating and functional. A miscarriage is preceded by fetal death, and presumably, death of the fetal blood cells. Thus, the answer to c may be “false” but this needs further study. False. Although not specifically discussed in the manuscript, fetal cell microchimerism also occurs in the rhesus monkey. False. Although there is evidence to suggest that HLA class II compatibility between a woman, her fetus, and her own mother plays a role in the development and maintenance of microchimerism, it is not required for the development of microchimerism.
27. (a) F
(b) T
(c) T
(d) F
(e) F
Explanation: (a) NSC have been identified throughout the early fetal brain. However in the adult mature brain NSC have been identified only in the subventricular zone
Appendix A19
(b)
(c)
(d) (e)
and the hippocampus. These areas support ongoing proliferation of neural stem cells that contribute to the generation of new neurones particularly following injury. Cells of neural morphology and phenotype have been derived from bone marrow and blood of human and rodents both in vitro and in vivo. This work is controversial, and several investigators have demonstrated that the apparent differentiation of bone-marrow to neural cells in vivo is actually caused by the fusion of a donor cell to a brain cell. NSC are highly proliferative and are commonly expanded as aggregates (neurospheres). Recently culture conditions have been established to grow them as monolayers. Importantly neurospheres do not just contain stem cells, but a population of stem cells with more differentiated progeny. NSC can generate the 3 major cell types of the CNS- neurones, oligodendrocytes and astrocytes. The other brain cell type, microglia, are derived from the blood. Although there are a variety of surface markers that enrich for NSC (eg Nestin, Musashi, Notch 1, CD133, etc) none is absolutely specific. The identification of NSC still relies on their ability to self renew and differentiate into neurones, oligodendrocytes and astrocytes.
28. (a) F
(b) F
(c) F
(d) T
(e) F
Explanation: (a) PVL is now infrequent (!2%) in extremely preterm infants. It is associated with a poor neurological prognosis. The commonest brain abnormality seen in preterm infants is diffuse increased signal intensity in the white matter on T2 weighted MR imaging. Although the pathological basis for this MR imaging finding is not confirmed. It is likely that it results from oligodendrocyte loss or injury. (b) Although HIE is a clear cause of cerebral palsy in some infants, the majority of infants do not have a clear birth insult although many have antenatal risk factors. (c) HIE typically leads to widespread cell loss with grey and white matter injury. Potential cell replacement strategies would need to consider this. (d) There are no neuroprotective treatments in widespread use for HIE at present. There are encouraging trials underway investigating the effect of brain cooling in such infants and the results awaited. In vivo studies suggest cooling reduces apoptotic cell death. (e) Most metabolic brain diseases are sporadic or autosomal recessive and so generally occur in previously unaffected families. If there is an affected sibling genetic/metabolic diagnosis may be possible antenatally. Many metabolic disorders have normal brain morphology and structure and so would not be diagnosed either with ultrasound or fetal MR. The majority of infants with metabolic brain diseases will present after the onset of symptoms.
A20 Appendix
29. (a) T
(b) F
(c) T
(d) T
(e) T
Explanation: (a)
(b)
(c)
(d)
(e)
The major advantage of perinatal cell therapy in metabolic disease is to intervene before considerable brain damage occurs. However the major challenge is to obtain an early accurate diagnosis. In addition NSC, unless genetically altered, may be vulnerable to the disease process itself, and even if successful would not alter systemic manifestations. There have been considerable advances in the genetic basis of metabolic diseases which aids diagnosis. However there remains a lack of knowledge of the cell and molecular interaction within the brain and the effects of the diseased brain environment. This knowledge would be necessary for the successful development of stem cell strategies. One would imagine the immature brain has more inherent plasticity and potential for repair, and it may well be that the environment of developing brain would contain the necessary cues for appropriate differentiation. However there is little systematic research studying the best developmental age for transplantation survival and integration. The immune system in fetal life and the early neonatal period is relatively naı¨ve. Again research is needed to support the assumption that grafting stem cells earlier may lead to improved graft tolerance and survival Undoubtedly the fetal or newborn brain is more accessible to direct injection of stem cells than the mature brain, but data suggests NSC are highly migratory and may actually home to sites of injury with simple injection into the CSF space. Also, as with gene therapy approaches there may be mechanical advantages to starting treatment in a smaller, rather than mature, brain in terms of the numbers of cells needed to populate a given area and instruct repair.
30. (a) T
(b) T
(c) F
(d) T
Explanation: (a) Commercially banked units are only for the use of the direct family and are unlikely to be used because the indications for BMT are rare (b) The commonest use for cord blood transplant is for acute leukaemia of childhood. This is a rare disease and is commonly cured by chemotherapy and transplant is rarely required (c) There are concerns that the cord blood of an infant who subsequently develops childhood leukaemia may contain markers of leukaemia predisposition. (d) Bacterial contamination with pathogens or low volume collections or clots in the collection are commonly found, especially if the person undertaking the collection is inexperienced.
Appendix A21
31. (a) T
(b) T
(c) F
(d) T
Explanation: (a) The midwife’s and obstetrician’s primary task is care of the infant/maternal pair (b) The interests of the cord collection may not best serve the interests of the donor infant (c) Bacterial contamination rates are a high as 30% in inexperienced hands but are reduced to less than 1% by dedicated trained staff, as demonstrated by the London Cord Blood Bank (d) The collection may be bacterially contaminated, of insufficient volume or contain large clots.