Stem Cells, Vol. 14, No. 3, 269-280,
May 1996
© 1996 AlphaMed Press
Hematopoietic Stem Cells in the Mouse Embryonic Yolk Sac
Robert Auerbach,
Hua Huang,
Lisheng Lu
Laboratory of Developmental Biology, University of Wisconsin, Madison, Wisconsin, USA
Key Words. Stem cell differentiation • Embryogenesis • Endothelial cells • Lineage restriction • Transplantation • Hemangioblasts
Dr. Robert Auerbach, Laboratory of Developmental Biology, University of Wisconsin, 1117 W. Johnson Street, Madison, WI, 53706, USA.
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Abstract
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The yolk sac is the first site of hematopoiesis during mammalian development. The yolk sac is also the first site of blood vessel development. Development of the blood islands in the yolk sac is an integrated process in which these two developmental events, hematopoiesis and vasculogenesis, proceed in concert. This review focuses on mouse yolk sac hematopoietic stem cells (YS-HSC), describing their differentiation in vitro and in vivo. YS-HSC go through a progressive series of changes prior to the initiation of lineage-specific differentiation. Experiments tracing their origins from postulated hemangioblasts, and the subsequent interaction between these stem cells and yolk sac endothelial cells are described. Differences between the extraembryonic YS-HSC and HSC found later within the embryo, perinatally or in adults, are described. YS-HSC have greater reproductive capability than HSC obtained from fetal liver, umbilical cord blood or adult bone marrow; they do not yet express major histocompatibility complex-associated antigens and they are able to reconstitute adult immunocompromised animals even when introduced in small numbers (<100 cells/mouse). With recent results demonstrating the feasibility of expanding YS-HSC in vitro as well as of introducing new genes into these cells by transfection, the YS-HSC shows promise both as a means of achieving long-term restitution of hematopoiesis across histocompatibility barriers and as a self-renewing vehicle for gene transfer.
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Introduction
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The term "hematopoietic stem cells" (HSC) almost always means stem cells found in the bone marrow, the site of replenishment of all lineages of blood cells in the adult. "Fetal HSC" generally refer to stem cells found in fetal bone marrow. The term "progenitor cells" also almost universally refers to cells from the bone marrow. In the majority of studies, progenitor cells are considered to be precursors of HSC, but in some publications the terms "progenitor cells" and "HSC" are used interchangeably.
A separate term, "embryonic stem cells" or "ES" cells, is used to describe the totipotent cells of the preimplantation blastocyst. These cells, prominent because of their use in the generation of transgenic animals, have the potential to develop into all lineages, including but not limited to the hematopoietic system.
With the terms "HSC" and "progenitor cells" preempted by the bone marrow, and "ES cells" by the blastocyst, we are left with inexact, often confusing terminology for the HSC derived from a large number of embryonic sites: the yolk sac, where hematopoiesis first takes place in development; the para-aortic region (aorta/gonad/mesonephros or AGM) within the early embryo; the embryonic and fetal liver; the fetal omentum; and the circulating blood of the fetal and perinatal (umbilical cord blood) period. Studies of the hematopoietic precursor cells derived from these several sources are increasing in number and import [1–6], and therefore it is necessary to use unambiguous terminology when discussing them.
In this review we discuss "HSC" that derive from the mammalian yolk sac. We will call them yolk sac HSC or YS-HSC throughout. We will describe our work and that of other research groups as it pertains to the ability of these cells to proliferate, migrate and differentiate in vitro as well as in vivo during embryogenesis, and upon transfer to adult host animals. Among the key questions to be answered are: whether the potential to differentiate into various lineages in vitro is an accurate reflection of what normally occurs during embryogenesis; to what extent the lineages generated by yolk sac precursor cells are similar to those generated by other hematopoietic precursor cells such as those from the embryonic or fetal liver, AGM or bone marrow; whether in vitro expansion of the hematopoietic precursors in the yolk sac can yield an increase in cells retaining the original characteristics of those stem cells; whether these cells—or their progeny—can repopulate adult, immunocompromised host animals to give rise to all or some subset of functionally competent mature blood cells; and whether on transfer into histoincompatible host animals these cells and their derived lineages can be maintained without rejection and thus provide long-term hematopoietic restitution.
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Heterogeneity of HSC
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Embryonic precursor cells of various origins such as the yolk sac, AGM or liver differ from each other as well as from the HSC of the bone marrow, although the final outcome of their differentiation—the generation of erythroid, lymphoid and myeloid lineages—may be similar. For example, the stem cells of the early murine yolk sac express no histocompatibility antigens (major histocompatibility complex [MHC] class I or II), stem cell antigen (Sca-1) or CD45 (Ly5), but express high levels of the adhesion molecule CD44, the heat stable antigen (HSA) and an epitope detected by antibody AA4.1. Stem cells of the fetal liver do express MHC class I and II, but at a lower level than stem cells in the bone marrow. Like yolk sac and unlike bone marrow, they have high levels of HSA and the AA4.1 antigen, but unlike yolk sac stem cells they express both CD45 and Sca-1 (Table 1).
The yolk sac is the earliest site of hematopoiesis in the mouse as it is in all amniotes [7]. Within the yolk sac, clusters of small cells become visible shortly after it first forms at day seven of mouse development. These clusters comprise the blood islands. Within 48-72 h, cells within the blood islands can be identified as primitive nucleated erythrocytes by their hemoglobin content. At about the same time, a vasculature develops and provides a visible means of blood circulation for the developing embryo (Fig. 1
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These two processes, the differentiation of erythrocytes and that of endothelial cells, occur within the yolk sac during normal development of the 9 to 10-day mouse embryo. What is the origin of these hemoglobin-producing primitive erythrocytes and of the vascular endothelial cells? Precursors of these cells must exist prior to embryonic day 9, with key questions being when and by what means these cells become committed to a distinct lineage. Early embryologists postulated that a single precursor cell can give rise to both endothelial cells and HSC [7]. The pattern of differentiation that they proposed was that a common precursor, the "hemangioblast," becomes committed to either the endothelial cell lineage (angioblast) or the hematopoietic lineages (HSC). It is the latter which is the subject of our review.
Within the yolk sac, as stated, embryonic (nucleated) red blood cells develop, the precursors of which appear well before they can be identified in nine-day-old embryos. What is most intriguing is that progenitor cells are present in the yolk sac at least as early as day 8 and probably even earlier, and are capable of differentiating into all other blood cell lineages, including non-nucleated erythrocytes, the many subsets of B and T cells, and the complete panel of definitive myeloid cells including granulocytes, megakaryocytes, mast cells and other cells of the macrophage/monocyte lineages. Despite this developmental potential, differentiation along these pathways does not normally proceed in the yolk sac [7]. Apparently these progenitor cells require a different microenvironment (e.g., thymus, liver) for differentiation into other than primitive erythrocytes or, alternatively, are inhibited from differentiation into the various leukocyte lineages by local factors produced within the yolk sac.
It now appears that there are several steps in stem cell differentiation within the yolk sac itself ([8, 9] and unpublished observations). We have defined these tentatively as Stage I yolk sac HSC (YS-HSC), Stage II YS-HSC and Stage III YS-HSC primarily on the basis of their cell surface phenotype (Table 1). What we now know is that there is the following: a YS-HSC in day 7-8 embryos that does not yet express either the early stem cell marker AA4.1, or the hematopoietic lineage marker CD45 (Ly5); a dominant YS-HSC in day 8-9 embryos, present until at least day 11, that expresses AA4.1, shows a weak expression of CD45, but does not express Sca-1 (Ly6); and a more "mature" stem cell (detected in vitro but only arguably in situ) that is similar to the earliest stem cell identified in the embryonic liver, in that it does express Sca-1 and detectable levels of MHC class I antigens (Table 1).
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Identification of Early Stem Cells in the Yolk Sac
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True multipotential stem cells are exceedingly sparse even in the youngest yolk sac studied to date (day 8) [21]. Phenotypic markers for these cells include:
a) the expression of a stem cell antigen identified by monoclonal antibody AA4.1 [11]. This antigen had previously been shown to be present on the multipotential stem cells in the midgestational (day 12-14) fetal liver [8, 9, 11, 13, 21, 22];
b) a relatively low buoyant density (<1.076) [11];
c) nonadherence, as defined by adhesion to plastic at 37°C [11]; and
d) the presence of a terminal sugar moiety identified by its ability to bind wheat germ agglutinin (WGA) [11].
Even within this small subset of cells, the true YS-HSC comprise a minority (2%) as measured by their ability to generate B cell and T cell lineages, as well as myeloid cells and erythrocytes. The majority of this subset of cells appears already committed to either erythropoiesis or myelopoiesis.
Recent studies, both in our laboratory and in others, indicate that there are HSC precursors at an even earlier stage of embryonic development (day 7.5-8.5). These cells have been identified as AA4.1–, but have been shown capable of giving rise to AA4.1+ cells given a suitable microenvironment and cytokine supplementation (unpublished observations).
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Lineage-Specific Differentiation
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There are two fundamentally different methods for analyzing the developmental propensity of YS-HSC. One involves the growth and differentiation of these cells in vitro, the other monitors the development of different cell lineages in vivo following transfer of stem cells into hematopoietically compromised host animals.
What we and others have found, however, is that conditions that lead to the differentiation of B cells, T cells, myeloid cells or erythrocytes are different for each cell lineage. For example, T cell differentiation can be obtained reliably only by providing a thymus organ microenvironment [11–13, 28–30]; B cell differentiation requires a stromal cell monolayer (generally the S17 cell line) [31, 32]; myeloid differentiation occurs best in a semisolid culture environment (generally methylcellulose) [33]; and erythropoiesis occurs best in cultures provided with erythropoietin as the principal growth factor [33]. Thus it has not been possible to test individual cells for totipotentiality prior to clonal proliferation.
T Cell Differentiation
Earlier studies have shown that cells from midgestational yolk sacs (day 11) could give rise to all the T cell lineages [12]. Liu and Auerbach [13] showed that as early as day 8, the yolk sac already contains T cell precursors. When provided with a stem cell-depleted thymus rudiment, yolk sac cells could enter the explant and differentiate to generate CD4+CD8+ Thy1+ lymphocytes, and subsequently could yield T cell receptor for antigen (TCR)
/
, TCR
/ß as well as definitive CD4+CD8– and CD4–CD8+ T cells [12, 13]. Recent studies by Dieterlen-Lievre and colleagues have obtained comparable results [9, 30, 34].
When we examined the minority of AA4.1+, WGA+ stem cells for their ability to generate T cells in the thymic rudiment microenvironment, we found that the T cell precursor frequency enrichment yielded a cell population that fully accounted for all of the T cell precursors in the early yolk sac ([11] and unpublished observations). A corollary to this finding is that in the absence of a suitable microenvironment in the yolk sac, lineage commitment to T cell differentiation does not appear to occur. This corollary was supported by the report that there exists in the thymic rudiment a small number of cells competent to develop into T cells that are not yet irreversibly committed to T cell differentiation [25]. It is reasonable to argue that it may be these cells that have migrated to the thymus from the yolk sac.
B Cell Differentiation
The finding that yolk sac cells can give rise to B cell lineages was first suggested by Tyan and Herzenberg [35] who reported that transplanted yolk sac cells, identifiable by an Ig allotypic difference from host cells, could generate Ig-producing cells when injected into stem cell-depleted irradiated host animals. More recent studies by Palacios and colleagues [36], Godin et al. [9] and in our own laboratory [10] confirmed that yolk sac nonadherent cells and, more specifically, yolk sac stem cells (AA4.1+, Lin–, Sca-1–), were able to generate cells in vitro that expressed cytoplasmic Ig, the B220 B cell-specific differentiation antigen and, subsequently, surface-associated Ig µ chain (s-IgM) when permitted to differentiate on S17 bone marrow stromal cell feeder layers or following transfer into stem cell-depleted (severe combined immunodeficient [SCID] or irradiated) mice. Furthermore, yolk sac stem cells yielded mature B cells in vivo following transfer into stem cell-depleted (SCID or irradiated) mice. Yolk sac-derived B cells could be detected more than six months after initial injection [10].
Myelopoiesis
A complete panel of myeloid cells has been generated from yolk sac cells grown under a variety of conditions in vitro [9, 10, 37] or following transplantation [7, 11, 15, 21, 38–40]. When enrichment procedures were used for obtaining yolk sac stem cells, 1/5 cells isolated were able to produce myeloid cells when grown under semisolid culture conditions [11]. The frequency of myeloid stem cell precursors was 10x that of precursors detected for either B or T cells, suggesting that many of the cells capable of myeloid cell differentiation were at a more lineage-committed stage than the most primitive, totipotent cells that could generate all lineages following clonal proliferation.
Erythropoiesis
As stated, the default mode for yolk sac hematopoiesis is erythropoiesis. The primitive erythrocytes, which are nucleated and express embryonic (or possibly fetal) hemoglobin [38, 41–43], can be produced in vitro under standard culture conditions. More efficient erythropoiesis occurs with addition of erythropoietin in combination with other growth-stimulating cytokines such as stem cell factor (SCF) and interleukin 3 (IL-3) [33].
Although the stem cell-enriched population identified by Huang and Auerbach is capable of erythropoiesis, and erythroid colonies are readily produced following addition of erythropoietin, the rapid increase in erythrocytes during days 9-11 of mouse development, i.e., during the yolk sac phase of erythropoiesis, suggests that there is a preponderance of cells in the early yolk sac that is already committed to the erythroid lineage.
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Clonal Expansion of HSC In Vitro
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To ascertain whether individual cells expressing the stem cell phenotypic markers (AA4.1+, WGA+, nonadherent, density <1.076) have the potential to give rise to all of the lineages, individual cells of this phenotype were distributed into single wells by cell sorting technology, where they were given appropriate substratum and growth factors for proliferation without promoting differentiation. Offspring from individual clones were tested variously for B cell, T cell and myeloid differentiation. We found that among the clones were some that could generate all three lineages (unpublished observations). Thus, the initial yolk sac cell was shown to be a true multipotential HSC. It is interesting that the cloning efficiency of the sorted cells conformed to the predictions from the previous calculations of precursor frequency, i.e., approximately 2% [10].
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Expansion of YS-HSC by Coculture with Yolk Sac Endothelial Cells
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AA4.1+, WGA+, low density nonadherent yolk sac cells from day 10 mouse embryos (Stage II YS-HSC) could be expanded >200-fold within eight days by providing them with a feeder layer of irradiated yolk sac-derived endothelial cells [14]. Both primary and secondary cultures could form mixed colonies without added growth factors and the colony-forming ability was retained for at least three passages in vitro. Retention of multipotentiality of the subcultured cells could be demonstrated by their ability to differentiate into lymphocytic (T and B) and myeloid (granulocytes, macrophages, monocytes, blast cells) lineages given appropriate culture conditions. Stem cell properties were maintained by a significant fraction of nonadherent cells in the third passage, although these stem cells expressed a somewhat more mature cell surface phenotype (AA4.1+, Sca-1+, HSA+, MHC class Iweak; i.e., Stage III YS-HSC) than the initial yolk sac stem cells (AA4.1+, Sca-1–, HSA–, MHC class I–). The fact that yolk sac endothelial cells could support the stable proliferation of single multipotential HSC in the absence of added growth factors supports the argument that the endothelium of the yolk sac is directly involved in the maintenance and differentiation of yolk sac stem cells.
It is not only our own work that has pointed to interaction between yolk sac endothelial cells and HSC. Williams and colleagues [44, 45] have found that SV-40 transformed endothelial cells can support bone marrow stem cell development, and L.A. Lasky has recently described experiments showing that myeloid and erythroid differentiation could be achieved utilizing a CD34+ YS-HSC/CD34+ yolk sac endothelial cell coculture system [46]. The interrelationship between endothelial cells and HSC in the yolk sac is also implied by two recent studies of transforming growth factor (TGF)-ß [47] and Flk-1 deficient [48] mice.
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In Vivo Reconstitution Using Yolk Sac Stem Cells
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Although the potential of yolk sac cells to generate various blood cell types following their injection into irradiated host animals has long been established, those early experiments required massive numbers of cells, leaving open the possibility that among these cells were some that had already achieved lineage restriction [7, 49]. The identification of a minority population that contained all of the multipotential stem cells made it possible to obtain more precise data on the ability of these stem cells to proliferate and differentiate in vivo.
When day 11 nonadherent, density <1.076, AA4.1+, WGA+ cells were injected into adult, syngeneic lethally irradiated mice, they generated B cells, T cells and myeloid cells [11, cf. 40] (markers to distinguish donor from recipient erythroid cells were not included in these experiments). However, the stem cells were not able to provide short-term protection from the lethal effects of irradiation, so that a radioprotective dose of bone marrow cells, appropriately marked to distinguish them from yolk sac cells, had to be administered concurrently. Yolk sac-derived leukocytes were first detectable after about three months and became the majority of leukocytes after six months.
Presumably, when yolk sac cells were introduced by intravenous injection into host animals, they were able to seed the appropriate organ sites where they could continue to proliferate and differentiate into the various lineages. Because yolk sac-derived mature T cells were detectable in the circulation, it may be assumed that some yolk sac cells went through several sequential developmental processes, first seeding the thymus, then differentiating in the thymus and then becoming peripheralized. It is likely that B cell differentiation was achieved in the spleen. The presence of CD5+ lymphocytes in the peritoneal cavity, moreover, suggests that the yolk sac contained not only the classic B cell precursors (B2 cells) but also the more primitive B1 cell precursors previously found only in the omentum and fetal liver [11].
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Response of Yolk Sac Stem Cells to Growth Factors
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HSC respond differentially to many different growth factors. This fact has prompted the use of complex media containing varying amounts of GM-CSF, M-CSF, IL-3, IL-6, IL-7, erythropoietin, TGF-ß and SCF, as well as the use of undefined growth supplements such as conditioned medium from stromal cell cultures and mixed leukocyte culture supernatants, in studies of hematopoiesis. Leukemia inhibitory factor and stem cell inhibitory factor have also been used, primarily in order to encourage proliferation of stem cells without concomitant lineage-restrictive differentiation.
We have recently examined the effects of some of these growth factors on purified yolk sac stem cells from day 11 embryos (Stage II YS-HSC; cf. Table 1 ). We found two special properties of yolk sac stem cells: 1) YS-HSC have an obligatory requirement for SCF, even in the presence of other growth factors, in contrast to stem cells from fetal liver or adult bone marrow (Fig. 2A). In the latter, SCF does have an effect when tested singly, but does not have an additive effect on stem cells provided with IL-3 and GM-CSF. 2) Yolk sac stem cells appear uniquely insensitive to TGF-ß. TGF-ß causes the loss of >80% of the AA4.1+, WGA+, nonadherent low density cell population, but leaves intact the primitive, high proliferative potential cells (Fig. 2B). Therefore, by combining SCF with TGF-ß it should be possible to generate large numbers of primitive yolk sac stem cells in vitro.
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Table 1. Comparision of HSC isolated from mouse embryonic yolk sac, day 7-11, day 11 fetal liver and adult bone marrow
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Figure 2. Effect of SCF (Fig. 2A ) and TGF-ß (Fig. 2B) on yolk sac (AA4.1+ WGAbright), fetal liver (AA4.1+, WGAbright) and bone marrow (Thy 1low, Lin–) stem cells. Yolk sac cells from day 11 embryos of C57BL6/Au mice were centrifuged on 1.077 Percoll for 25 min to eliminate erythrocytes and high density cells, then incubated in plastic dishes for one h. The relatively nonadherent cells were then panned by immunocytoadherence to AA4.1 antibody. Finally AA4.1+ yolk sac cells were labeled with fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (WGAFITC ) and sorted flow cytometrically for the bright population [6]. AA4.1 + fetal liver cells [3, 18] were obtained from 14-day C57BL6/Au embryos in similar fashion. Bone marrow cells were first depleted of CD4, CD8 and RB6-8C5 (granulocyte) positive cells by magnetic separation. The remaining cells were stained for Thy1.2 FITC and positive cells were selected by magnetic separation. The Thy1 positive cells were stained for Mac1 (M1/70) and B220 (14.8) using a second-step rat IgGPE reagent. Thy1lo Lin–cells were sorted on the basis of intermediate forward scatter (FSC), high side scatter (SSC), intermediate level of fluorescein staining (Thylo) and low level of phycoerythrin staining (Lin–). 80%-90% of sorted bone marrow cells had "blast"-like morphology. The isolated cells were seeded 500-2500 per 35 mm petri dish in Dulbecco's modified Eagle's medium containing 30% fetal bovine serum, 1% bovine serum albumin, 10–4M 2ME and 0.8% methylcellulose. Growth factors were added as indicated in the figure. Cultures were incubated in 5% CO2 fully humidified incubator at 37°C for seven days. Colonies were counted under an inverted microscope, and numbers were normalized against GM-CSF plus IL-3 for each experiment. Purified human platelet-derived TGF-ß 1 was a generous gift from Dr. Anita Roberts (Bethesda, MD).
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Expression of Receptor Tyrosine Kinases on Yolk Sac Stem Cells
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Recently there has been a surge in the number of studies identifying receptor tyrosine kinases on various embryonic stem cells. As shown in Tables 1 and 2, a large number of receptor tyrosine kinases are expressed on purified YS-HSC [50–52]. Marked differences can be seen between the kinases found at different times during development of the yolk sac (e.g., day 8 versus day 11). For example, Huang and Lemischka [19] found that a kinase sharing a high percentage of homology to a yeast SNF and tie-2 was present on day 8 yolk sac stem cells but absent on day 11 (Table 1). flt-4 and c-kit were most abundant on day 8, while c-fms was expressed predominantly on day 11. The expression of tyrosine kinases in YS-HSC also differed from tyrosine kinases reported to be present in fetal liver or adult bone marrow [19, 22–27]. Although data have not been obtained for the AA4.1– Stage I YS-HSC, it seems reasonable to assume that they would express at least those receptor tyrosine kinases present on both ES cells and Stage II YS-HSC (e.g., FGFr and flk-1).
Many of the receptor tyrosine kinases present on primitive YS-HSC are also present on the early yolk sac angioblasts. Indeed, flk-1, the first "fetal liver kinase", has frequently been identified as "specific" to endothelial cells. Similarly, tie and tek, previously identified on primitive yolk sac vascular endothelial cells, are also found expressed in day 8 YS-HSC. Where the branch point between endothelial cell precursors and HSC occurs is not known.
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Hemangioblasts: Evidence for a Common Precursor for HSC and Endothelial Cells
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We have pointed out earlier that the primitive mammalian yolk sac contains both hematopoietic precursors and precursors of vascular endothelial cells, the former giving rise to all the blood cell lineages while the latter are responsible for the formation of the embryonic vasculature. More than a century ago it was proposed that a single progenitor cell, the "hemangioblast", could diverge to give rise to these two distinct pathways. Until recently there were no experimental observations that addressed this question [cf.53]. Recent experimental results, however, refocus attention on this postulated common progenitor cell.
What is of particular interest is the fact that when adherent cells from day 8 yolk sacs were placed under appropriate growth conditions, they developed along two separate pathways (unpublished observations). These early adherent cell cultures were able to generate nonadherent AA4.1+ cells while maintaining an adherent AA4.1– cell population. The adherent cells could differentiate into endothelial-like cells when provided with growth factors such as endothelial cell growth supplement, bFGF, heparin and tumor-conditioned culture supernatant (manuscript in preparation).
Although cloning of the initial adherent cell isolates has not yet been achieved, these results are consistent with the concept of a single hemangioblast precursor cell within the yolk sac which diverges to give rise to angioblasts and HSC. Because, as stated, both endothelial cell precursors and HSC express flk-1, a receptor tyrosine kinase important for endothelial cell differentiation (by binding of vascular cell adhesion molecule), as well as a key kinase involved in HSC differentiation, we predict that flk-1 will be present on hemangioblasts. On the other hand, it is likely that lineage-specific markers such as CD45 for hematopoietic cells or CD31 for endothelial cells will not be present on the postulated hemangioblasts.
The origin of hemangioblasts is still not resolved. It is not clear whether they have a dual origin (the intraembryonic AGM and the extraembryonic yolk sac), whether they originate intraembryonically and migrate to the yolk sac, or whether they are initially found in the yolk sac and migrate into the embryo. Recently, Dieterlen-Lievre and colleagues [9] showed that HSC arise simultaneously in the yolk sac and the AGM. Much emphasis has recently been placed on this intraembryonic source of HSC [9, 54, 55, 30, 40, 50, 56]. However, interpretation of the AGM results is complicated by the fact that primordial germ cells, which are localized to this region, have the potential to generate hematopoietic progenitor cells [57–59] as, indeed, do embryonic stem cells [60–63].
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Yolk Sac Stem Cell Transplantation: Prospects and Problems
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As stated earlier, one of the most significant differences between HSC from the yolk sac and those found subsequently in fetal liver or bone marrow is the apparent lack of expression of MHC class I and MHC class II antigens. At the same time they have the greatest potential for replication.
With recent results demonstrating the feasibility of expanding YS-HSC in vitro [14] as well as of introducing new genes into these cells by transfection [29, 64, 65], YS-HSC must be viewed as promising candidates both for achieving long-term restitution of hematopoiesis across histocompatibility barriers and, as well, for use as self-renewing vehicles for gene transfer.
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Acknowledgments
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We appreciate the help provided by Wanda Auerbach throughout the writing of this manuscript, including both skilled bibliographical retrieval and editorial assistance. We thank David Auerbach for the preparation of Figure 1. Original research included in this paper was supported by grants EY3243 and HL52148 from the National Institutes of Health, as well as by a gift from Progenitor, Inc., Columbus, Ohio.
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Footnotes
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Note added in proof:
The following publication appeared after submission of this manuscript, and describes the work cited as reference 49: Fennie C, Cheng J, Dowbenko D et al. CD34+ endothelial cell lines derived from murine yolk sac induce the proliferation and differentiation of yolk sac CD34+ hematopoietic progenitors.
Blood 1995;12:4454–4467.
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accepted for publication January 22, 1996.
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Abstract
Introduction
