Stem Cell Amplification Research Articles

A Simplified Procedure for Hematopoietic Stem Cell Amplification Using a Serum-Free, Feeder Cell-Free Culture System

Umbilical cord blood (UCB) is increasingly being used as a donor source of hematopoietic stem cells (HSCs) to treat blood malignancies. The main limitation to the widespread use of UCB is the low number of HSCs per unit. To compensate, a strategy of in vitro stem cell amplification has been attempted in different research laboratories. The major hurdle blocking success is the creation of culture conditions that support the growth of hematopoietic stem cells without their differentiation. We have designed a simple culture system for stem and progenitor cell expansion that resulted in an increased number of hematopoietic stem cells that maintain their ability to home to the bone marrow and to permanently engraft.

The stem cell niche of human livers: Symmetry between development and regeneration

Human livers contain two pluripotent progenitors: hepatic stem cells and hepatoblasts. The hepatic stem cells uniquely express the combination of epithelial cell adhesion molecule (EpCAM), neural cell adhesion molecule (NCAM), cytokeratin (CK) 19, albumin +/-, and are negative for alpha-fetoprotein (AFP). They are precursors to hepatoblasts, which differ from hepatic stem cells in size, morphology, and in expressing the combination of EpCAM, intercellular cell adhesion molecule (ICAM-1), CK19, albumin++, and AFP++. The hepatic stem cells are located in vivo in stem cell niches: the ductal plates in fetal and neonatal livers and canals of Hering in pediatric and adult livers. The hepatoblasts are contiguous to the niches, decline in numbers with age, wax and wane in numbers with injury responses, and are proposed to be the liver's transit-amplifying cells. In adult livers, intermediates between hepatic stem cells and hepatoblasts and between hepatoblasts and adult parenchyma are observed. Amplification of one or both pluripotent cell subpopulations can occur in diseases; for example, hepatic stem cell amplification occurs in mild forms of liver failure, and hepatoblast amplification occurs in forms of cirrhosis. Liver is, therefore, similar to other tissues in that regenerative processes in postnatal tissues parallel those occurring in development and involve populations of stem cells and progenitor cells that can be identified by anatomic, antigenic, and biochemical profiles.

Stem cells for regenerative medicine—Biological attributes and clinical application

The place of stem cells in the therapeutic armamentarium is currently subject to intense scrutiny. One issue is the nature of the stem cells themselves, and their relationship to better-known stem cells, such as hematopoietic stem cells. Here it is argued that the stem cells we propose for therapeutic application may be more closely related to embryonic cells than are hematopoietic stem cells. A second issue is how the stem cells may best be manipulated for therapeutic use; this includes stem cell purification, amplification, fate selection, and suitability for clinical use.

Oriented immobilization of epidermal growth factor onto culture substrates for the selective expansion of neural stem cells

To develop a culture substrate that allows efficient expansion of neural stem cells (NSCs), epidermal growth factor (EGF) was immobilized onto the Ni(II)-chelated surface of a glass-based substrate through coordination of Ni(II) to the histidine tag that was fused to the C-terminal of EGF using recombinant technology. For the preparation of the nickel-chelated surface, a thin gold layer was deposited to the glass surface, and then the self-assembled monolayer of alkanethiol terminated with trivalent carboxylic acids was formed on gold and chelated with Ni(II) ions. In the preparation of a monolayer, triethylene glycol-terminated alkanethiol was mixed with carboxylic acid-terminated alkanethiol at various compositions in order to reduce the non-specific adsorption of EGF. The surface analysis of the monolayers was performed by X-ray photoelectron spectroscopy, infrared reflection–absorption spectroscopy, and contact angle measurements. Surface plasmon resonance analyses and protein assays were performed for characterizing EGF-immobilized surfaces. The proliferation and differentiation of rat fetal NSCs were examined on the EGF-chelated substrates to assess quantitatively the effects of alkanethiol composition on the efficiency of stem cell amplification. It was shown that the amplification efficiency was dependent on the alkanethiol composition. This result could be attributed to the difference in the surface density of chelated EGF. Under the optimal condition, 98% of proliferated cells expressed NSC marker. In addition, these cells could be subcultured for further expansion, while retained their multipotency. We concluded that the substrate developed here provides the efficient method for the highly selective expansion of NSCs.

Overexpression of integrin-linked kinase induces cardiac stem cell expansion

Recent evidence suggests that the adult heart contains stem cells that are capable of self-renewal as well as multilineage differentiation. However, their inherent capacity for self-renewal is limiting to cell replacement applications. Integrin-linked kinase is a multifunctional protein kinase that activates Wnt target genes implicated in the symmetric replication of embryonic stem cells. Primary cultures derived from human fetal cardiac tissue (19-22 weeks' gestation) were grown in serum-free media and evaluated for the presence of cardiac progenitor cells. The effect of integrin-linked kinase was ascertained by adenoviral overexpression. Cultures infected with wild-type integrin-linked kinase yielded a significant (P = .001), approximately 5-fold increase in both the absolute number and the frequency of c-Kit-positive, myosin-negative cells. Cardiospheres, comprised on morphologically homogeneous, anchorage-independent cells, were reproducibly present at days 7 to 10 and formed derivative cardiospheres in multiple passages. Integrin-linked kinase infection of primary cardiac cell cultures resulted in a greater number of primary spheres at each cell density tested, compared with untreated and virus controls (P = .001). Secondary spheres transferred to differentiation medium and 5-aza-deoxycytodine (10 micromol/L) generated cells exhibiting biochemical evidence of differentiation into cardiomyocytes, smooth muscle cells, and endothelial cells. This study demonstrates that self-renewing cardiospheres generated from human fetal cardiac cells are composed of cells exhibiting the properties of stem cells, including the capacity for self-renewal and multilineage differentiation. Our results suggest that integrin-linked kinase promotes stem cell amplification and can be applied therapeutically to overcome a major limitation in the field of cardiac regenerative medicine.

Evidence for Restricted Glycolytic Metabolism in Primary CD133+ Cells.

The slow cycling, location and hypoxia-resistance of hematopoietic stem cells are suggestive of a restricted metabolism. We propose that HSC metabolism is adapted to unique metabolic conditions supplied by the stem cell niche, and that a combination of the metabolic and signalling environments acts to support stem cell amplification and to limit it to a narrowly-defined and physiologically rare set of sites. To investigate this possibility, we have established moderate throughput, small scale cultures to examine the metabolic characteristics of primary CD133+ cells isolated from umbilical cord blood. A screen of carbon and energy sources revealed that pyruvate (but neither fatty acids nor amino acids) can replace glutamine as a major substrate. The fact that pyruvate contributes significantly to the cellular metabolism even in the presence of glucose suggests that CD133+ cells employ an unusually low level of glycolysis. Flow cytometric analysis of surface markers before and after culture confirmed that the addition of glucose (0mM, 5mM and 25mM) or insulin (0μg/mL, 4 μg/mL) increased the overall cell yield, but had no effect on the proliferation of early cells (CD133+, CD34+ and c-kit+). In parallel, metabolic profiling of undifferentiated and differentiated FDCPmix cells using gas chromatography and mass spectrometry techniques revealed an accumulation of glucose in the self-renewing (undifferentiated) population. Taken together, these observations suggest that glycolysis makes little contribution to stem cell metabolism, and that hematopoietic stem cells (as has been suggested for germ cells) may instead use glycolytic products supplied by stromal cells. Furthermore, our studies have revealed an unexpected effect of osmolarity on both glucose metabolism and self-renewal, in that an increase in osmolarity from 0,32 Osm/Kg to 0,36 Osm/kg reduced the rate of glucose-dependent proliferation of CD133+ cells without reducing the yield of early (CD133+ CD34+, ckit+) cells. Similarly, both the proportion of self-renewing cells in FDCPmix cultures and the recruitment of these cells to active self-renewal in semi-solid media in a colony forming assay were found to be increased at higher osmolarity. This suggests that high osmolarities suppreses both glycolytic metabolism and proliferation rate, but favour the maintenance of “early” progenitors. Finally, we have applied our assay to test a range of different growth factor combinations (of SCF, Flt3-ligand, TPO, IL3, IL6, IL7, IL11 and TGF-b) in 18% and 1% O2, and found that hypoxia extends markedly the range and magnitude of the proliferation response. Taken together, our results suggest that hematopoietic stem cells are indeed adapted to a rare metabolic microenvironment which includes (but is probably not limited to) low oxygen and glucose concentrations, and that the metabolic environment is likely to influence strongly the response to growth factors. A thorough understanding of stem cell metabolism may therefore provide a basis for more controlled manipulation of HSC in vitro.

Hematopoietic Stem Cell Transduction and Amplification in Large Animal Models

Progress in retroviral gene transfer to large animal hematopoietic stem cells (HSCs) has led to efficient, reproducible long-term marking in both canine and nonhuman primate models. Successes for HSC gene therapy have occurred in the severe combined immunodeficiency setting, in which transduced cells have a selective advantage. However, for most diseases, the therapeutic transgene does not confer a sufficient survival advantage, and increasing the percentage of gene-marked cells in vivo will be necessary to observe a therapeutic effect. In vivo amplification should expand the potential of HSC gene therapy, and progress in this area has benefited greatly from the use of large animal models where efficacy and toxicity have often not correlated with results in murine models. To date, the best results have been observed with O(6)-methylguanine-DNA methyltransferase (MGMT) selection, with which increases in gene-marked repopulating cells have been maintained long-term, likely because of the toxicity of 1,3-bis-(2-chloroethyl)-1-nitrosourea and temozolomide to quiescent HSCs. Using MGMT selection, long-term marking levels exceeding 50% can now be routinely attained with minimal toxicity. There is cause to be optimistic that HSC gene therapy with in vivo amplification will soon allow the treatment of several genetic and infectious diseases.

Hematopoietic Stem Cell Transduction and Amplification in Large Animal Models

Hematopoietic Stem Cell Transduction and Amplification in Large Animal Models

Endogenous Myc controls mammalian epidermal cell size, hyperproliferation, endoreplication and stem cell amplification

The transcription factor Myc (c-Myc) plays an important role in cell growth and cell death, yet its physiological function remains unclear. Ectopic activation of Myc has been recently suggested to regulate cell mass, and Drosophila dmyc controls cellular growth and size independently of cell division. By contrast, it has been proposed that in mammals Myc controls cell division and cell number. To gain insights into this debate we have specifically knocked out Myc in epidermis. Myc epidermal knockout mice are viable and their keratinocytes continue to cycle, but they display severe skin defects. The skin is tight and fragile, tears off in areas of mechanical friction and displays impaired wound healing. Steady-state epidermis is thinner, with loss of the proliferative compartment and premature differentiation. Remarkably, keratinocyte cell size, growth and endoreplication are reduced, and stem cell amplification is compromised. The results provide new and direct evidence for a role for endogenous Myc in cellular growth that is required for hyperproliferative cycles and tissue homeostasis.

Regulation of Primitive Hematopoietic Cell Growth and Differentiation by the Gata-2 Transcription Factor.

The GATA-2 transcription factor is required for the development and regulation of blood stem cells as judged by the embryonic lethal, “bloodless” phenotype of mice lacking GATA-2 and the high levels of GATA-2 present in adult stem cells which necessarily decline with differentiation into the various blood lineages. Our previous studies showing that sustained, high level GATA-2 expression in adult murine stem cells blocks both stem cell amplification and differentiation in vivo (Persons, Blood 93:488, 1999) suggest that the fate of primitive repopulating cells is regulated by the level of GATA-2. To further investigate the stem cell regulatory activity of GATA-2, we developed a retroviral vector encoding a tamoxifen inducible, GATA-2/estrogen receptor chimeric protein (G2-ER). In the presence of tamoxifen, murine hematopoietic progenitors transduced with the G2-ER/GFP retroviral vector generated only 6% ± 5% (n=5) of the number of myeloid colonies formed in the absence of tamoxifen, consistent with our previous findings and confirming the functional activity of the fusion protein. Surprisingly, in the absence of tamoxifen, G2-ER colonies grew significantly larger in size than control colonies. In addition, G2-ER transduced bone marrow (BM) cells out-competed mock-transduced cells in liquid culture. Control GFP or G2-ER transduced BM cells were diluted with mock-transduced BM cells at a 1:10 ratio. At 7 days, the control culture was 10% GFP+ and the G2-ER culture was 17% positive. However, by day 11 the control culture remained 10% GFP+, while the G2-ER culture increased to 87% GFP+. Thus far, G2-ER transduced cells can be expanded for up to 12 weeks in culture and, in contrast to control cultures which are morphologically comprised of mast cells after the same time in culture, the G2-ER cells are predominantly immature appearing myeloid cells and blasts. FACS analysis confirmed the myeloid phenotype of the expanded G2-ER cells (83% CD11b+, c-kit−), while the control cells showed a mast cell phenotype (100% c-kit+, IgE receptor+). The G2-ER pro-proliferative effect was also observed in transplanted mice since there was a significant repopulation advantage of G2-ER myelo-erythroid cells over untransduced cells. Mice transplanted with G2-ER transduced BM mixed 1:1 with untransduced marrow at 12 weeks post-transplant showed 82% ± 6 GFP+ Gr-1+ cells (n=10) and 57% ± 4 RBCs compared to 28% ± 17 (n=7) and 10% ± 5 in GFP: Mock transplanted control mice. These differences were not due to differences in the transduction rate of primitive repopulating cells as judged by gene transfer into the Sca+ Lin− cells contained in the grafts. Interestingly, there was a lack of B lymphoid development from transduced stem cells in G2-ER transplanted mice at 12 weeks since few peripheral blood B220+ cells co-expressed GFP (8% ± 2), compared to controls (58% ± 14). Similarly, development of transduced T cells was also delayed but showed a rebound with low expressing GFP+ cells at late time points. Analysis of bone marrow, spleen, and thymus of transplanted mice confirmed the poor development of lymphoid cells and showed that there was not a developmental arrest in these organs. Since it is likely that small amounts of G2-ER are active in the absence of tamoxifen, these data suggest that the concentration of GATA-2 in specific lineage precursors is critical in regulating the expansion and differentiation into the different blood lineages. Studies to identify GATA-2 interactions with other transcription factors may uncover the mechanisms that regulate the differentiation of specific lineage precursors.

55. Alteration of Primitive Hematopoietic Cell Fate and Growth by the GATA-2 Transcription Factor

The GATA-2 transcription factor is required for the development and regulation of blood stem cells as judged by the lethal, “bloodless” phenotype of mice lacking GATA-2 and the high levels of GATA-2 present in adult stem cells which necessarily decline with differentiation into the various blood lineages. This idea is consistent with our previous studies showing that sustained, high level GATA-2 expression in adult murine stem cells blocks both stem cell amplification and differentiation in vivo (Persons, Blood 93:488, 1999). Recent studies implicate GATA-2 in maintaining p21 and p27 proteins (Ezoe, Blood 100:3512,2002), thereby contributing to the quiescence of the stem cell pool. In order to determine if p21 and p27 mediate this role in hematopoietic stem cells (HSCs) in vivo, a GATA-2 retroviral vector was used to enforce expression in p21/p27 null HSCs. Although Sca+Lin− p21/p27 null cells were efficiently transduced with both the GATA-2 and control vectors, engraftment of transduced cells was only observed with the control vector. These data suggest that GATA-2 regulated stem cell quiescence is independent of p21/p27. To further investigate the stem cell regulatory activity of GATA-2, we developed a retroviral vector encoding a GATA-2/estrogen receptor chimeric protein (G2-ERT2). Hematopoietic progenitors transduced with the retroviral G2-ERT2/GFP vector generated few GFP+ colonies in the presence of tamoxifen, relative to the number formed in the absence of tamoxifen (6 ± 5%, n = 5), consistent with our previous findings. In the absence of tamoxifen, G2-ERT2 colonies grew significantly larger in size. In addition, G2-ERT2 transduced bone marrow (BM) cells out-competed mock transduced cells in liquid culture. Control GFP or G2-ERT2 transduced total BM cells were diluted with mock transduced BM cells at a 1:10 ratio. At 7 days, the control culture was 10% GFP+ and the G2-ERT2 culture was 17% positive. However, by day 11 the control culture remained 10% GFP+, while the G2-ERT2 culture increased to 87% GFP+. In contrast to control cultures, the G2-ERT2 cultures were predominantly composed of immature-appearing cells. This G2-ERT2 pro-proliferative effect was also observed in vivo. We found a significant repopulation advantage of G2-ERT2 myelo-erythroid cells over untransduced cells in mice transplanted with G2-ERT2 transduced BM. Mice transplanted with G2-ERT2 transduced BM mixed 1:1 with untransduced marrow at 12 weeks showed 82% ± 6 GFP+ Gr-1+ cells (n = 10) and 57% ± 4 RBCs compared to 28% ± 17 (n = 7) and 10% ± 5 in GFP:Mock control mice. Interestingly, there was a lack of lymphoid development since few B220+ cells co-expressed GFP in G2-ERT2 transplanted mice at 12 weeks (8% ± 2), compared to controls (58% ± 14). Since it is likely that small amounts of G2-ERT2 are active in the absence of tamoxifen, the concentration of GATA-2 is likely critical in normally regulating primitive hematopoietic cell function and fate decisions. Thus, constitutive, low concentrations of GATA-2 lead to a pro-proliferative effect in myeloid cells but inhibit lymphoid development. Since our data suggest that GATA-2 does not mediate its effects through the p21/p27 pathway, investigation of GATA-2 interactions with other transcription factors and their effects on gene regulation is needed.

Deregulated expression of HOXB4 enhances the primitive growth activity of human hematopoietic cells

AbstractIdentification of the molecular mechanisms that can promote human hematopoietic stem cell amplification is a major goal in experimental and clinical hematology. Recent data indicate that a variety of regulatory molecules active in early development may also play a role in the maintenance of hematopoietic stem cells with repopulating activity. One important class of early developmental genes determining hematopoietic development are homeobox transcription factors. Here, we report that retrovirally mediated expression of the homeobox geneHOXB4 rapidly triggers an increase in the number of human hematopoietic cord blood cells with stem cell and progenitor cell properties detected both by in vitro and in vivo assays. This growth enhancement extended across primitive myeloid-erythroid and B-lymphoid progenitors but did not lead to alterations in the balance of lymphomyeloid reconstitution in vivo, suggesting that HOXB4does not affect control of end-cell output. These findings revealHOXB4 as a novel, positive regulator of the primitive growth activity of human hematopoietic progenitor cells and underline the relevance of early developmental factors for stem cell fate decisions.

Enforced P-glycoprotein pump function in murine bone marrow cells results in expansion of side population stem cells in vitro and repopulating cells in vivo

AbstractThe human multidrug resistance-1 (MDR1) gene product, P-glycoprotein (P-gp), is well known for its ability to confer drug resistance; however, recent evidence suggests that P-gp expression can have more general effects on cellular development. In support of this idea, it was previously shown that retroviral-mediated MDR1 expression in murine bone marrow cells resulted in the expansion of stem cells in culture and in the development of a myeloproliferative syndrome in transplanted mice. It is now reported that MDR1-mediated stem cell expansion is associated with an increase in side population (SP) stem cells, defined by Hoechst dye staining. Transduction of murine bone marrow cells with an MDR1 retroviral vector resulted in an almost 2 log increase in SP cell numbers over 12 days in culture, whereas there was a rapid loss of SP cells from control cultures. Stem cell amplification was not limited to ex vivo expansion cultures but was also evident when MDR1-transduced cells were directly transplanted into irradiated mice. In these cases, stem cell expansion was associated with relatively high vector copy numbers in stem cell clones. As previously reported, some cases were associated with a characteristic myeloproliferative syndrome. A functionally inactive MDR1 mutant cDNA was used to show that P-gp pump function was required both for amplification of phenotypically defined SP cells and functionally defined repopulating cells. These studies further support the concept that ABC transporter function can have important effects on hematopoietic stem cell development.

Enforced P-glycoprotein pump function in murine bone marrow cells results in expansion of side population stem cells in vitro and repopulating cells in vivo

The human multidrug resistance-1 (MDR1) gene product, P-glycoprotein (P-gp), is well known for its ability to confer drug resistance; however, recent evidence suggests that P-gp expression can have more general effects on cellular development. In support of this idea, it was previously shown that retroviral-mediated MDR1 expression in murine bone marrow cells resulted in the expansion of stem cells in culture and in the development of a myeloproliferative syndrome in transplanted mice. It is now reported that MDR1-mediated stem cell expansion is associated with an increase in side population (SP) stem cells, defined by Hoechst dye staining. Transduction of murine bone marrow cells with an MDR1 retroviral vector resulted in an almost 2 log increase in SP cell numbers over 12 days in culture, whereas there was a rapid loss of SP cells from control cultures. Stem cell amplification was not limited to ex vivo expansion cultures but was also evident when MDR1-transduced cells were directly transplanted into irradiated mice. In these cases, stem cell expansion was associated with relatively high vector copy numbers in stem cell clones. As previously reported, some cases were associated with a characteristic myeloproliferative syndrome. A functionally inactive MDR1 mutant cDNA was used to show that P-gp pump function was required both for amplification of phenotypically defined SP cells and functionally defined repopulating cells. These studies further support the concept that ABC transporter function can have important effects on hematopoietic stem cell development.

Regulation of DNA replication during the cell cycle: roles of Cdc7 kinase and coupling of replication, recombination, and repair in response to replication fork arrest.

DNA replication is central to cell growth, development, and generation of tissues and organs. Recent advances in understanding replication machinery have revealed striking conservation of components involved in the processes of DNA replication, from yeasts to human. The conservation extends even to bacteria for some basic components of replication apparatus. Eukaryotic DNA replication is regulated at various stages to ensure strict regulation during cell cycle. We have identified a novel mammalian kinase, Cdc7-ASK (Activator of S phase Kinase), that plays a key role at the entry into S phase as a molecular switch for DNA replication. This kinase is specifically activated during S phase and triggers the firing of DNA replication by phosphorylating an essential DNA helicase component of the replication complex. Environmental stresses such as DNA damages or depletion of essential nutrients for DNA synthesis lead to unscheduled arrest of DNA replication forks. In bacteria, this leads to induction of altered modes of DNA replication, which may repair DNA damages, facilitate reassembly of replication machinery at the stalled replication fork, or do both. In eukaryotes, blocking replication forks usually induces both checkpoint responses, which prevent premature progression of cell cycle events before precise completion of the preceding cell cycle stage, and the recombinational repair system for the lesions. Possible common bases in recognition of stalled replication forks in bacteria and eukaryotes will be discussed. Furthermore, we will discuss the potential of replication and checkpoint proteins as targets of anticancer agents as well as possible novel technology for stem cell amplification through manipulation of DNA replication.

A genetically myeloablated MPS VII model detects the expansion and curative properties of as few as 100 enriched murine stem cells

Causes of transplantation failures are often difficult to assess due to our inability to monitor hematopoietic stem cell (HSC) homing, distribution, and amplification in situ. We have developed a mouse model that permits histochemical localization of 1000-fold enriched HSC and quantification of their long-term expanded progeny in situ. The mice are genetically myeloablated ( c-kit receptor mutated, W 41/ W 41) and are β-glucuronidase null (GUSB − ; gus mps/ gus mps). The GUSB − mice with mucopolysaccharidosis type VII (MPS VII), like a large number of human patients with similar diseases, have systemic lysosomal storage disease that leads to premature death. Congenic GUSB + , Lineage lo, Sca-1 hi, c-Kit hi, Hoechst lo HSC, at doses of 30, 100, 250, and 425 cells, implanted and amplified in adult W 41/ W 41, gus mps/ gus mps recipients in a dose-dependent manner. At autopsy, primary recipients of 100 and 425 donor cells had histologically identifiable donor GUSB + cells in multiple sites and showed both myeloid and lymphoid expansion in bone marrow. Donor cells were rare in the liver and spleen of 100-cell recipients, but lysosomal storage was significantly reduced. The life span was significantly extended in engrafted recipients of 250 (36.7 ± 3.84 weeks, p = 0.0316 ) and 425 (40.7 ± 1.53 weeks, p = 0.0033 ) cells compared to untreated mice (26.4 ± 1.53 weeks). Secondary hosts of marrow from the recipients of 425 cells demonstrated continued expansion of the GUSB + cells. Results indicate the genetically myeloablated MPS VII mice can be used to trace and enumerate donor cells long-term and to follow early engraftment events in situ.

Haematopoietic stem cell emergence and development in the human embryo and fetus; perspectives for blood cell therapies in utero

In this chapter, we review the successive stages of haematopoietic development in humans. Primitive haematopoiesis does not take place in the <i>embryo proper</i>, but in the yolk sac, where the first haematopoietic cells are observed as early as 19 days, forming blood islands of CD34⁺ cells closely associated with adjacent CD34⁺ endothelial cells. Recent observations have indicated that definitive haematopoiesis occurs first in the 1-month embryo, where numerous clustered CD34⁺ haematopoietic cells have been identified in the ventral aspect of the dorsal aorta; these aorta-associated cell clusters may reflect unique phases of stem cell induction and amplification. Active blood formation is observed in the liver and bone marrow by the end of the first trimester. Inception of haematopoeisis occurs, however, earlier in the liver, where CD34⁺ cells are detected as early as 23 days, than in the marrow, where haematopoietic cells are not observed before week 11. T-lymphopoiesis develops from 9 weeks onwards, in the thymus colonized by circulating stem cells. Stem cells from fetal liver and marrow differ from adult marrow stem cells by a number of features, the more significant one being a higher cycling rate. The relative paucity of functional T cells during the second trimester, the considerable proliferative capacity of fetal liver stem cells and the lack of functional T cells in fetal stem cell harvests prompted some investigators to perform in utero transplantations during the second trimester using 7–14-week-old fetal liver stem cells. The procedure is not without risk of fetal death. However, clear-cut success has been obtained. Two children with complete correction of their initial immunodeficiency disease are alive and in good condition 9 and 10 years after in utero transplantation. Studies <i>in vitro</i> have confirmed the complete immunological tolerance between donor-derived T cells and host-derived B cells and monocytes, confirming the validity of the hypotheses at the basis of in utero grafting. Better understanding of fetal stem cell emergence and behaviour and of the fetal haemopoietic microenvironment should permit improvement in the time of stem cell collection and allow <i>ex vivo</i> expansion before in utero transplantation, which should broaden the indications of this promising treatment.

Evidence of Both Ontogeny and Transplant Dose-Regulated Expansion of Hematopoietic Stem Cells In Vivo

Recent assessment of the long-term repopulating activity of defined subsets of hematopoietic cells has offered new insights into the characteristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their self-renewal in vivo. We have now exploited the ability to quantitate these cells using the competitive repopulating unit (CRU) assay to identify the role of both intrinsic (ontological) and extrinsic (transplanted dose-related) variables that may contribute to the regulation of CRU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liver (FL) or the bone marrow (BM) of adult mice injected 4 days previously with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-spleen numbers irrespective of the number or origin of cells initially transplanted. In contrast, regeneration of Ly5.1+ donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult BM. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in recipients of FL and adult BM transplantation, respectively, with an accompanying maximum CRU amplification of 150-fold for recipients of FL cells and 15-fold for recipients of adult BM cells. Interestingly, the extent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulatory mechanisms to prematurely limit stem cell self-renewal ability. Proviral integration analysis of mice receiving retrovirally transduced BM cells confirmed regeneration of totipotent lymphomyeloid repopulating cells and provided evidence for a greater than 300-fold clonal amplification of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sources that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell amplification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage for attempts to enhance CRU regeneration by administration of agents that may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic regenerative capacity.