In Utero Transplantation of Expanded Autologous Amniotic Fluid Stem Cells Results in Long‐Term Hematopoietic Engraftment

Abstract In utero transplantation (IUT) of hematopoietic stem cells (HSCs) has been proposed as a strategy for the prenatal treatment of congenital hematological diseases. However, levels of long‐term hematopoietic engraftment achieved in experimental IUT to date are subtherapeutic, likely due to host fetal HSCs outcompeting their bone marrow (BM)‐derived donor equivalents for space in the hematopoietic compartment. In the present study, we demonstrate that amniotic fluid stem cells (AFSCs; c‐Kit+/Lin−) have hematopoietic characteristics and, thanks to their fetal origin, favorable proliferation kinetics in vitro and in vivo, which are maintained when the cells are expanded. IUT of autologous/congenic freshly isolated or cultured AFSCs resulted in stable multilineage hematopoietic engraftment, far higher to that achieved with BM‐HSCs. Intravascular IUT of allogenic AFSCs was not successful as recently reported after intraperitoneal IUT. Herein, we demonstrated that this likely due to a failure of timely homing of donor cells to the host fetal thymus resulted in lack of tolerance induction and rejection. This study reveals that intravascular IUT leads to a remarkable hematopoietic engraftment of AFSCs in the setting of autologous/congenic IUT, and confirms the requirement for induction of central tolerance for allogenic IUT to be successful. Autologous, gene‐engineered, and in vitro expanded AFSCs could be used as a stem cell/gene therapy platform for the in utero treatment of inherited disorders of hematopoiesis. stem cells 2019;37:1176–1188


INTRODUCTION
In utero transplantation (IUT) of hematopoietic stem cells (HSCs) is a potential nonmyeloablative strategy for the treatment of many hematologic disorders [1,2]. Engraftment of allogenic HSCs requires both induction of donor-specific immunologic tolerance through thymic processing of donor antigen [3][4][5][6] and successful competition of donor HSCs for receptive niches in the robust, nonmyeloablated fetal hematopoietic compartment [7]. Failure of either component results in ultimate failure of engraftment. Experimentally, engraftment of donor cells after IUT can be accomplished across full major histocompatibility complex (MHC) barriers under specific circumstances in both murine [8,9] and large animal models [10][11][12]. In the murine model of allogenic IUT, we and others have shown that successful engraftment of adult bone marrow (BM) cells requires effective delivery of a relatively large dose of cells within a specific gestational age time window to achieve tolerance [13][14][15]. On the contrary, highly enriched HSCs do not engraft even in high doses [3], suggesting that, in the BM, there are cell populations other than the HSCs required for successful antigen presentation in the fetal thymus. The developmental status of donor cells also influences engraftment with fewer fetal liverderived cells being required than adult BM, presumably due to the greater competitive capacity of fetal HSCs [9,16]. However, fetal liver cells are difficult to obtain, autologous transplantation would not be possible, and their isolation from aborted fetuses has raised challenging ethical questions. Although we previously documented an adaptive immune response in recipients of IUT that were nonchimeric [14], this immune response was subsequently demonstrated to be secondary to a maternal alloresponse after IUT that was passed via allo-antibodies in breast milk to induce the adaptive response in the pups [17]. In pups placed on a foster (naïve) mother after allogenic IUT, all of the recipients engraft and there is no evidence of an allo-immune response [17].
In this context, we wished to assess the hematopoietic potential of a promising source of donor cells, the amniotic fluid stem cell (AFSC) [18], in the well-characterized murine model of IUT. In the last few years, we have explored the hematopoietic potential of C-Kit+ (CD117+)/Lineage− (Lin−) AFSCs [19]. Freshly isolated murine AFSCs have a phenotype similar to fetal liver (FL) HSCs [20] and can engraft after postnatal transplantation into adult immunocompromised hosts [19]. We have also recently demonstrated that intraperitoneal IUT of AFSCs generates multilineage hematopoietic engraftment [21]. Similarly, freshly isolated CD34+ sheep AFSCs can be genetically modified overnight and are able to engraft after IUT in the hematopoietic system in an autologous setting [22]. AFSCs have several potential advantages that may be salutary for IUT. They can be easily derived from routine amniocentesis, which is commonly performed for prenatal diagnosis of genetic disorders, and early second trimester collection would allow expansion in time for autologous treatment of the target disease using a stem cell/gene therapy approach [20,23]. Their fetal source and high proliferative capacity in vivo may allow them to more effectively compete with endogenous FL-HSCs resulting in higher levels of engraftment compared with adult-derived cells. Moreover, they can be efficiently genetically modified allowing the potential for an autologous cell-gene therapy or gene editing approach [18,22,24]. However, their therapeutic utility has been so far limited because their hematopoietic potential was lost after in vitro expansion using "mesenchymal-type" culture protocols (culture in adherence with serum rich media) [18,20]. Here, we demonstrate for the first time that expansion of AFSCs on feeder layers of mitotically inactivated embryonic fibroblasts using "embryonic-type" culture media maintains their hematopoietic characteristics and results in highly efficient engraftment of in the congenic murine model of IUT. Conversely, no engraftment was seen in the allogenic setting suggesting inadequate presentation of donor antigen in the developing/fetal thymus. These findings support the potential use of in vitro-expanded, autologous AFSCs for the treatment of hematopoietic disorders.

Animals
Time-dated C57BL/6J (B6; H2Kb+) or Balb/c (H2Kd+) pregnant mice were used as fetal IUT recipients at 14 days of gestation (E14; Fig. 2A). AFSCs were isolated at E13 from C57BL/6TgN (act-enhanced green fluorescent protein) OsbY01 (B6-green fluorescent protein [GFP]) or Balb/c (H2Kd+) fetuses. For congenic IUT, B6-GFP AFSCs were transplanted in B6 fetuses ( Fig. 2A). For allogenic IUT, Balb/c AFSCs were transplanted in B6 fetuses ( Fig. 2A), or B6-GFP AFSCs were transplanted in Balb/c fetuses. Experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children's Hospital of Philadelphia and University College London (PPL number: 70/7408), and followed guidelines set forth in the NIH Guide for the Care and Use of Laboratory animals, as well as the Animals (Scientific Procedures) Act (1986) and the NC3Rs Animal Research: Reporting of In Vivo Experiments guidelines [25].
Freshly isolated AFSCs were cultured as described previously (see Supporting Information Methods) [26]. Cell growth/ proliferation was quantified at 1, 3, 6, and 8 days of culture using an MTS-based colorimetric assay (MTS Cell Proliferation Assay; Abcam, Cambridge, Massachusetts, US). After 8 days, cells were harvested and sorted for CD117 (Supporting Information Table S1)

AFSC Characterization
Purity, Cell Surface Marker Expression, and Cell Cycle Analysis The purity of MACS-isolated AFSCs was evaluated by FACS using a CD117 antibody (Supporting Information Table S1). Fresh and cultured CD117+ AFSCs were further characterized by assessing expression of CD45, Sca-1, CD34, and H2Kb (Supporting Information Table S1). In order to identify the proportion of freshly isolated AFSCs that were in one of the three interphase stages of the cell cycle (G0/G1, S, G2/M), we used a propidium iodide (PI; Sigma, St. Louis, Missouri, United States)based protocol. Fresh AFSCs were fixed in 70% ethanol, treated with ribonuclease A, and stained with PI (400 μl of 1 mg/ml stock solution per 10 6 cells) prior to cell cycle analysis by FACS.

Hematopoietic Gene Expression Analysis
Single cell quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of AFSCs was performed as described previously (see Supporting Information Methods) [27]. We focused our analysis on the key hematopoietic regulators www.StemCells.com To further characterize the expression of hematopoietic genes in fresh and cultured AFSCs, we used a commercially available gene array (RT 2 Profiler PCR Array; PAMM-054Z; Qiagen, Hilden, Germany) according to manufacturer's instructions (see Supporting Information Methods). Expression of these genes in AFSCs was compared with that observed in BM-HSCs. Data analysis was performed using online software (http:// pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php).

Colony Forming Assay
The in vitro hematopoietic potential of fresh and cultured AFSCs was evaluated using colony forming assays. A total of 10 4 cells (fresh or cultured CD117+ AFSCs) were added to 2.5 ml of semisolid media (Methocult; Stem Cell Technologies Canada Inc., Vancouver, U.K.), plated in 35 mm dishes, and cultured in a humidified incubator at 37 C and 5% CO 2 . Colonies were characterized and counted after 14 days of culture.

IUT
Intravenous IUT was performed at E14 as previously described (see Supporting Information Methods) [14,15]. Half of the surviving injected offspring in the allogenic group were nursed at birth by naive (noninjected) surrogate mothers in order to exclude transfer of maternal allo-antibodies through breast milk [17].

Donor Cell Tracking in the Fetus
In order to determine donor cell distribution in recipients following IUT, fetuses were harvested at E14 (4 hours post-IUT), E17, and E19. Fetuses were dissected under a microscope and key hematopoietic tissues including the fetal liver, spleen, and thymus were exposed. Qualitative assessment of the presence of donor cells in these tissues was performed by detecting GFP+ cells with a fluorescent microscope.

Donor Cell Hematopoietic Engraftment and Multilineage Differentiation
Chimerism was assessed in peripheral blood, BM, spleen, and thymus from E14 injected mice at 4 (postnatal day 28, P28), 12 (P84), and 24 (P168) weeks of age. MNCs were stained with an antibody against CD45 (Supporting Information Table S1). In experiments using B6-GFP donor cells, engraftment was assessed as the percentage of CD45+ cells that were GFP+ by FACS, with chimerism being defined as more than 1% GFP+. In experiments using Balb/c donor cells, MNCs were stained with a H2Kd antibody (Supporting Information Table S1), and the percentage of CD45+ cells that were H2Kd+ was determined by FACS, with chimerism being defined as more than 1% H2Kd+. Qualitative confirmation of engraftment results obtained by FACS was obtained using immunohistochemistry (BM, spleen thymus collected at P28; see Supporting Information Methods). Multilineage differentiation of donor cells was assessed by FACS in donor-derived MNCs in blood and BM at 24 weeks using antibodies against lymphoid (CD3, B220), myeloid (CD11b, Gr-1), and erythroid (BM only; Ter119) markers (Supporting Information Table S1).

Adaptive Immune Response Assessment
Spleen and lymph nodes were harvested from B6 (H2Kb+) mothers and their progeny that received allogenic (Balb/c; H2Kd+) AFSCs IUT (with or without fostering) at 4 weeks of age. The cellular adaptive immune response was assessed using the in vivo mixed lymphocyte reaction (MLR) as described previously (see Supporting Information Methods) [17]. The frequency of alloreactive T cells in vivo was quantified as described by Suchin et al. [28].
The humoral adaptive immune response was assessed using serum from 4-week-old allogenic IUT recipient B6 mice and Balb/c splenocytes as described previously (see Supporting Information Methods for allo-antibody assay) [17].

Statistical Analysis
Data were compared using unpaired Student's t test, one-way or two-way analysis of variance (see Supporting Information Methods for details).

IUT of Allogenic AFSC Results in an Adaptive Immune Response and Donor Cell Rejection
To elucidate a possible role of an effector T-cell response in the lack of chimerism after IUT of allogenic AFSCs, we performed in vivo MLR [28]. The relative frequency of alloreactive T cells at 72 hours was 19.9% AE 1.0% for allogenic and 22.9% AE 0.9% for allogenic-fostered animals (Fig. 3A, 3B). These values were significantly higher than the ones observed in naïve B6 mice (mice that did not receive IUT; 12.4% AE 0.5%; p < .001 versus allogenic and allogenic-fostered), but similar to those seen in B6 mice immunized with Balb/c cells (21.5% AE 0.4%; Fig. 3B). Similar analysis in mothers of allogenic AFSCs recipients demonstrated lack of sensitization of the maternal immune system (Fig. 3C).
Serum from allogenic and allogenic-fostered pups demonstrated significantly higher median anti-IgG fluorescence intensity compared with negative controls (naïve) at serum-totarget cell ratios of 1:10, 1:2, and 1:1 (similar to that observed in immunized animals; Fig. 3D). This is consistent with antidonor allo-antibody formation in pups that received IUT of allogenic AFSCs regardless of fostering. As was the case for alloreactive T cells, there was no evidence of a maternal humoral response to IUT of allogenic AFSCs (Fig. 3E).

Lack of Thymic Homing Explains Rejection of Allogenic AFSC Following IUT
In utero tracking experiments were used to investigate the rejection and lack of engraftment of allogenic AFSCs. In order to be able to detect allogenic AFSCs easily in fetal tissues, we performed IUT of B6-GFP cells in Balb/c donors. Donor cell tracking was performed at: (a) E14 (4 hours post-IUT) to assess homing of AFSCs to the fetal liver and thymus, (b) E17 to evaluate migration of cells to the fetal spleen, and determine whether donor cells could be found in the thymus prior to closure of the "thymic window" for development of central (deletional) tolerance, and (c) E19 to assess donor cell distribution immediately before birth (Fig. 4A, 4B). Both congenic and allogenic AFSCs (10 4 per fetus) homed to the fetal liver soon after IUT, proliferated, and migrated to the fetal spleen with no obvious differences detected between groups at any analysis time point (Fig. 4A). Following this, we focused our attention on the thymus and compared homing of AFSCs to that of allogenic BM-MNCs (10 7 B6-GFP BM-MNCs per Balb/c fetus that result in stable hematopoietic engraftment across immune barriers post-IUT [17], or 10 4 per fetus to control for cell number), and that of allogenic BM-HSCs (CD117+, Sca-1+, Lin−; 10 4 B6-GFP BM-HSCs per Balb/c fetus as stem cell control group). A significant number of donor (GFP+) cells were detected in the thymus post-IUT of either 10 4 or 10 7 BM-MNCs from as early as 4 hours following transplantation (Fig. 4B). This was in contrast to what we observed for both congenic and allogenic AFSCs, which, despite engrafting in other hematopoietic organs, did not home to the thymus at any time point (Fig. 4B). Surprisingly, when BM-HSCs were used for IUT, donor cells were not seen in the thymus until E19 (Fig. 4B), which raised questions about the ability of such an enriched donor cell population to induce tolerance in this setting.
To address this issue, we assessed engraftment at 4, 12, and 24 weeks of offspring age in all experimental groups included in our tracking analysis. As we have reported previously [14], IUT of congenic and allogenic BM-MNCs resulted in macrochimerism in blood with no significant differences detected between the two groups in any of the analysis time points (Fig. 4C). In contrast, IUT of congenic but not allogenic AFSCs resulted in stable, long-term hematopoietic macrochimerism in 100% of transplanted animals (Fig. 4C), with significantly higher levels of engraftment achieved in congenic AFSCs animals compared with recipients of congenic or allogenic BM-MNCs (p < .001; Fig. 4C). The lack of engraftment of B6-GFP AFSCs following IUT in Balb/c fetuses is similar to what we observed when Balb/c AFSCs were transplanted in B6 fetuses (Fig. 2C, 2D). No engraftment was observed when enriched allogenic BM-HSCs were used, with only microchimerism achieving post-IUT of congenic BM-HSCs (Fig. 4C). As was the case with allogenic AFSCs (Fig. 3), there was evidence of an adaptive immune response in allogenic BM-HSCs recipients resulting in rejection of donor cells (Supporting Information Fig. S4A, S4B). These findings confirm the requirement for timely (prior to E17) donor cell homing to the thymus for induction of central/deletional tolerance and explain the paradoxical rejection and lack of engraftment of allogenic AFSCs.

AFSC Can Be Expanded Without Losing Their Hematopoietic Potential
Having established the remarkable hematopoietic capacity of AFSCs post-IUT, we tested their in vitro expansion potential. CD117+/Lin− AFSCs were successfully cultured on a feeder layer of mitotically inactivated MEF (Fig. 5A) [26]. The present culture protocol allows expansion without loss of CD117 expression following 8 days of culture (CD117+; cultured AFSCs: 84.3% AE 0.9%; Fig. 5B). Further characterization showed that CD45 expression was also maintained (90.4% AE 9.5%; Fig. 5C), with levels of expression of other hematopoietic markers raised compared with freshly isolated AFSCs, and low levels of MHC class I expression (Fig. 5C). Almost all cells remained negative for non-CD45 hematopoietic lineage markers (Lin−: 0.6% AE 0.2%). Hematopoietic gene array analysis of cultured AFSCs and comparison with BM-HSCs showed similar levels of expression in 73.5% (61/83) of examined genes, with significant upregulation in only 1.2% (1/83) and downregulation in 22.9% (19/83) of examined genes ( Fig. 5E and Supporting Information Fig. S1B, S1D, S1F). Direct comparison of hematopoietic gene expression in fresh and cultured AFSCs showed similar levels of expression in 90.5% (74/84) of examined genes (Fig. 5D). Single cell qRT-PCR demonstrated expression of the hematopoietic regulators Lmo2 (56%), Gata1 (35%), and Gata2 (21%; Fig. 5F and Supporting Information Fig. S2A). We also looked into expression of pluripotency regulators and found that although c-Myc and Klf4 expression was maintained in cultured AFSCs compared with fresh (65% and 58%, respectively), Oct4 expression was significantly reduced (13%; Supporting Information Fig. S2C). Similar to the fresh AFSCs, only 4% of analyzed cultured AFSCs expressed Sox2, and none of the cells expressed Nanog (Supporting Information Fig. S2C). Microscopic analysis of cultured AFSCs showed minimal proliferation for the first 3 days, followed by significant growth thereafter. To assess cell growth formally, CD117+ cells were isolated and counted at day 8 of culture, demonstrating a fourfold rise in the number of cells over the 8-day culture period (fold-increase: 4.1 AE 0.3). These findings were confirmed with MTS-based colorimetric analysis (Fig. 5G), and are consistent with AFSCs expansion. Finally, cultured AFSCs demonstrated in vitro hematopoietic function in semisolid media (Fig. 5H, 5I) similar to that of fresh AFSCs. Using this culture system, we have managed to expand murine AFSCs for up to five passages (cell passage when 80% confluence achieved, typically after 6-8 days of culture) without any effect on cell surface phenotype or in vitro hematopoietic potential.

IUT of Cultured AFSC Results in Long-Term, Multilineage Hematopoietic Engraftment
Our final in vivo study determined the ability of fresh and cultured AFSCs to engraft the hematopoietic system long-term post-IUT. We compared hematopoietic chimerism achieved with fresh and cultured AFSCs for up to 24 weeks post-IUT (Fig. 6A). Fetal survival was 61.9% in the cultured AFSCs group, similar to fresh AFSCs recipients (Fig. 6B). Hematopoietic macrochimerism was achieved in 100% of animals that received fresh or cultured AFSCs, with similar levels of engraftment in blood observed in the two experimental groups at 4 weeks of age (P28; fresh AFSCs: 21.9% AE 1.0%; cultured AFSCs: 21.3% AE 1.2%; Fig. 6C, 6D). Hematopoietic engraftment was stable in fresh and cultured AFSCs recipients at 12 (P84) and 24 weeks of age (P168) with no differences observed between groups (Fig. 6D). Moreover, multilineage analysis of donor cells in recipient blood at 24 weeks of age (P168; Fig. 6E) demonstrated normal differentiation to lymphoid (CD3+, B220+) and myeloid (CD11b+, Gr-1+) lineages similar to that observed in the host (Fig. 6E, 6F).

DISCUSSION
We demonstrate here that IUT of autologous cultured AFSCs resulted in long-term, multilineage hematopoietic engraftment. On the contrary, IUT of allogenic AFSCs led to an adaptive immune response and rejection most likely related to the lack of thymic homing of donor cells during gestation.
C-Kit+/Lin− AFSCs isolated from mouse amniotic fluid showed hematopoietic characteristics with expression of hematopoietic markers and key hematopoietic regulators. Most AFSCs were in quiescence, in keeping with a primitive HSCs phenotype, which is also evident when their genetic profile is compared with more mature BM-HSCs. AFSCs exhibited significant clonogenic potential, producing colonies from all three hematopoietic lineages when cultured in semisolid media. This is in keeping with what we have previously demonstrated [19,21], and further highlights the potential of obtaining fetal HSCs from an easily accessible source during gestation [20]. Most importantly, we www.StemCells.com ©2019 The Authors. STEM CELLS published by proved here that following 8 days of culture and a fourfold rise in the number of CD117+ cells, AFSCs maintained a similar phenotype and exhibited significant hematopoietic potential equal to that of freshly isolated AFSCs (similar numbers of hematopoietic colonies were observed when identical numbers of fresh and cultured AFSCs were placed in semisolid media). Remarkably, both freshly isolated and expanded congenic AFSCs showed high engraftment potential in vivo. In particular, 100% of animals receiving intravenous IUT of congenic 10 4 fresh or cultured AFSCs showed stable hematopoietic macrochimerism for up to 6 months, with engraftment in blood and BM exceeding 20%. This remarkable engraftment potential may also be related to the intravenous injection technique which is superior to previously described intraperitoneal delivery and is more applicable to possible clinical translation [13][14][15]. This is also demonstrated by our recently published preliminary study in which intraperitoneal IUT of 10 4 congenic AFSCs per fetus resulted in long-term hematopoietic engraftment only of around 10%, which is approximately half to that achieved in the present study using intravenous IUT [21]. Multilineage long-term engraftment equivalent to that observed in the host was confirmed in the recipient groups. Indeed, given the relatively small number of AFSCs in the amniotic fluid, clinical application in the setting of IUT for the treatment of hematological disorders will require adequate expansion without loss of their hematopoietic characteristics. This finding opens-up the possibility of using AFSCs for the treatment of congenital hematological or other inherited disorders [18,19,23,26]. In particular, it would be advantageous to combine cell and gene therapy with the subsequent correction and expansion of AFSCs, which can be given back to the same fetus with the aim of correcting hematological or other monogenic diseases before birth [22]. Clinically applicable ultrasound guided IUT of transduced autologous AFSCs has already been achieved in fetal sheep with a low complication rate [22,29]. Both cord blood and BM-derived HSCs have shown the ability for limited expansion maintaining their hematopoietic potential [30][31][32][33]. However, the methods described did not work with AFSCs expansion, leading to inefficient replication and loss of their potential. In keeping in with their more primitive phenotype, we adopted here a method described previously to expand pluripotent stem cells, and we have observed a remarkable proliferative potential of the AFSCs while maintaining both in vitro and in vivo hematopoietic potential.
Having established the high engraftment potential of AFSCs, we sought to explore further the possibility of using allogenic AFSCs. This approach could be advantageous for congenital diseases because (in combination with in vitro expansion) it would allow banking of clinical-grade, cost-efficient cell lines, and would preclude the need for genetic correction [20]. However, we confirmed with this study that intravenous IUT leads to minimal engraftment of allogeneic AFSCs in hematopoietic tissues (similar to intraperitoneal IUT; 21). These unexpected results were in contrast to what we and others reported for allogeneic BM-MNCs [17] as well as fetal liver-derived MNCs [34] injected in utero, and led us to investigate different mechanisms which could have resulted in the rejection of allogenic AFSCs post-IUT. When considered together, our alloreactive Tcell and allo-antibody data confirm our recently published preliminary findings [21], and are consistent with an adaptive immune response in allogenic AFSCs recipient pups resulting in rejection of donor cells. Although separate third-party antigen control experiments were not performed, the results obtained from naïve (negative control) and immunized (positive control) groups in MLR and allo-antibody experiments confirm direct immunization as the cause of allogenic AFSCs rejection. Moreover, fostering of newborns in the allogenic group with noninjected surrogate mothers to negate any maternal immune response had no effect on chimerism, which is also in contrast to what has been reported previously using allogenic BM-MNCs [17]. Further studies confirmed that there was no evidence of a maternal cellular or humoral response to IUT of allogenic AFSCs. The absence of sensitization of the maternal immune system and lack of effect of fostering on engraftment suggest that our findings are due to the failure of induction of central/deletional tolerance in the offspring and not due to maternal factors [17], although we did not investigate placental trafficking of maternal cells that has been previously shown to result in loss of hematopoietic chimerism post-IUT [34]. These results were somewhat paradoxical and might be related to the more immature phenotype of ASFC, or the comparatively small cell number used for IUT. Finally, the present experimental series provides further mechanistic insight for the rejection of donor AFSCs following allogenic IUT. We here demonstrate that, both for AFSCs and BM-HSCs, there is a requirement for timely (likely prior to E17; BM-HSCs and/or their progeny home to the thymus but not until E19) donor cell homing to the thymus for induction of central/deletional tolerance [4][5][6]. The lack of timely migration of the AFSCs or their progeny to the thymus may explain their rejection and lack of engraftment.
One of the limitations of our study relates to the strains of mice used in the present experimental series. Allogenic IUT was performed using Balb/c AFSCs transplanted into B6 recipients and congenic IUT utilized B6-GFP cells administered in B6 mice. Thus, some of our findings could simply be due to strain differences in the AFSCs themselves or to decreased sensitivity of the detection method in the allogeneic strain combination. Although we did not perform a formal comparison of Balb/c and B6-GFP-derived AFSCs, we have characterized freshly isolated AFSCs from a variety of mouse strains (including Balb/c; Supporting Information Fig. S3) and shown similar cell surface phenotype as well as in vitro hematopoietic potential [21]. Moreover, lack of engraftment of allogenic AFSCs was also observed when B6-GFP were transplanted in Balb/c fetal recipients (Fig. 4), suggesting that our findings are due to rejection of allogenic donor cells and not due to strain-related differences and/or methodological issues. In addition, the lack of tolerance induction post-IUT of allogenic AFSCs could be due to the inability (rather than delay) of these cells to generate progeny able to present antigen to the thymus. However, this is unlikely based on the in vitro and in vivo multilineage hematopoietic potential of these cells, as well as the adaptive immune response-mediated rejection of allogenic BM-HSCs (which, by default, have the ability to generate antigenpresenting cells). It is possible that a specific "tolerance-inducing cell" must be cotransplanted with stem cells (AFSCs, BM-HSCs, or otherwise) in order for allogenic IUT to be successful; in vitro generation of such cells is likely to require special (differentiation-inducing) culture conditions, as AFSCs-derived antigen presenting cells do not seem to be a by-product of our culture system (although we did not look formally, the vast majority of expanded cells are cKit+ and hematopoietic lineage negative). The latter, in addition to the time-course of rejection of AFSCs following allogenic IUT, is under investigation in our laboratory. Finally, the findings of the present experimental series may be limited to the specific subpopulation of amniotic fluid-derived stem cells. Although prenatal therapies targeting diseases of hematopoiesis would utilize cKit+/Lin− AFSCs, cKit− amniotic fluid-derived mesenchymal stem cells (AFMSC) could be utilized for in utero treatment of inherited disorders such as osteogenesis imperfecta [20,23,35]. Allogenic IUT may be feasible in the latter setting, as the well-recognized immunomodulatory properties of MSC (including AFMSC; [36]) may facilitate successful engraftment across immune barriers [20,23,35].
Clinical application of AFSCs in the setting of IUT would necessitate demonstration that human AFSCs can be expanded in the laboratory without losing their hematopoietic potential, and have similar characteristics following prenatal transplantation. Amniocentesis clinical samples obtainable from 15 weeks of gestation contain approximately 1% AFSCs (1,000-2,000 CD117+ AFSCs in 2 ml amniotic fluid). Extrapolating from murine IUT results, we estimate that at least 12 population doublings will be required for sufficient IUT at 20 weeks of gestation (fetal weight 300 g, 40,000 AFSCs/g). Using congenic AFSCs allows IUT at a later time point (20 weeks gestation versus 14 weeks when allogenic cells are used; the latter is equivalent to E17 in mice), which is both clinically desirable (due to the potential complications of IUT during early gestation) and provides a 5-week period for AFSCs expansion and gene engineering. Identification of the optimal human AFSCs expansion conditions while maintaining desirable cellular characteristics is currently a focus of investigation in our laboratory. Gene engineering of autologous AFSCs may have immunological implications following IUT, as it will ultimately lead to introduction of "foreign" protein (e.g., α or β globins) after successful engraftment of autologous cells in hematopoietic niches and subsequent maturation/lineage commitment. The latter may have significant implications on efficacy of the proposed therapeutic approach. In the setting of congenic/autologouslike murine IUT, the presence of "foreign" protein (required to detect donor cells in immunologically matched congenic hosts; e.g., GFP in the current series of experiments, or CD45.1 in previously published work from out laboratory [21]) did not affect engraftment or multilineage differentiation of donor cells with no evidence of an adaptive immune response. Despite these reassuring findings and given the limitations of murine IUT models, we and others are utilizing animal models of prenatal transplantation that resemble human IUT more closely in order to address this and other important issues prior to clinical application.

CONCLUSION
Our findings suggest that both freshly isolated and expanded AFSCs could be used as an autologous stem cell source for the prenatal treatment of congenital hematological disorders. This would require sampling of amniotic fluid with gene correction and subsequent expansion of AFSCs before selection and IUT via intravenous injection at the optimal gestational age for engraftment and antigen presentation to the fetal thymus. In fact, our study demonstrates far greater hematopoietic potential in the fetal environment for a given cell dose of AFSCs compared with adult BM-derived MNCs, as well as enriched BM-HSCs, although formal limiting dilution studies were not performed. However, in contrast to congenic (autologous-like) AFSCs, allogenic AFSCs did not engraft because of lack of induction of tolerance in the fetus. The latter is related to the inability of AFSCs or their progeny to migrate to the thymus for timely presentation of donor antigen. These findings reveal the remarkable hematopoietic potential of AFSCs in the setting of autologous/congenic IUT, and confirm the requirement for induction of central tolerance for allogenic IUT to be successful.