The human chorion contains definitive hematopoietic stem cells from the fifteenth week of gestation.

We examined the contribution of the fetal membranes, amnion and chorion, to human embryonic and fetal hematopoiesis. A population of cells displaying a hematopoietic progenitor phenotype (CD34++ CD45low) of fetal origin was present in the chorion at all gestational ages, associated with stromal cells or near blood vessels, but was absent in the amnion. Prior to 15 weeks of gestation, these cells lacked hematopoietic in vivo engraftment potential. Differences in the chemokine receptor and ß1 integrin expression profiles of progenitors between the first and second trimesters suggest that these cells had gestationally regulated responses to homing signals and/or adhesion mechanisms that influenced their ability to colonize the stem cell niche. Definitive hematopoietic stem cells, capable of multilineage and long-term reconstitution when transplanted in immunodeficient mice, were present in the chorion from 15-24 weeks gestation, but were absent at term. The second trimester cells also engrafted secondary recipients in serial transplantation experiments. Thus, the human chorion contains functionally mature hematopoietic stem cells at mid-gestation.

Maternal decidual macrophages inhibit NK cell killing of invasive cytotrophoblasts during human pregnancy.

Human pregnancy is an immunological paradox. Semiallogeneic (fetal) placental cells (extravillous cytotrophoblasts [CTBs]) invade the uterine lining (decidua), which contains a unique decidual natural killer (dNK) cell population, identified by the cell surface phenotype CD56(bright) CD16(-) CD3(-) and CD14(+) CD206(+) macrophages (dMac). Previous reports suggested that human dNK cells are not a threat to the fetoplacental unit because they are anergic. In contrast, here we showed that purified and exogenously stimulated dNK cells are capable killers of cellular targets, including semiallogeneic CTBs. However, dMacs in the decidual leukocyte (DL) population restrained dNK killing through a transforming growth factor beta1 (TGF-beta1)-dependent mechanism. Our findings support a new model whereby dNK cells, capable of killing CTBs, are prevented from doing so by neighboring macrophages, thus protecting the fetal cells from NK cell attack. We speculate that this mechanism would inhibit dNK cell-mediated killing, even under conditions where high levels of cytokines may stimulate dNK cells, which could pose a threat to the developing placenta.

Human placenta and chorion: potential additional sources of hematopoietic stem cells for transplantation.

BACKGROUND: Hematopoietic stem cell (HSC) transplantation is an essential element of medical therapy, leading to cures of previously incurable hematological and nonhematological diseases. Many patients do not find matched donors in a timely manner, which has driven efforts to find alternative pools of transplantable HSCs. The use of umbilical cord blood (UCB) as a source of transplantable HSCs began more than two decades ago. However, the use of UCB as a reliable source of HSCs for transplantation still faces crucial challenges: the number of HSCs present in a unit of UCB is usually sufficient for younger children but not for adults, and the persistent delayed engraftment often seen can result in high rates of infection and mortality. STUDY DESIGN AND METHODS: We propose a new approach to a solution of these problems: a potential increase of the limited number of UCB-HSCs available by harvesting HSCs contained in the placenta and the fetal chorionic membrane available at birth. RESULTS: We investigated the presence of hematopoietic progenitors and HSCs in human placenta and chorion at different gestational ages. The characterization of these cells was performed by flow cytometry and immunolocalization, and their functional status was investigated by transplanting them into immunodeficient mice. CONCLUSION: HSCs are present in extraembryonic tissues and could be banked in conjunction to the UCB-HSCs. This novel approach could have a large impact on the field of HSC banking and, more crucially, on the outcome of patients undergoing this treatment by greatly improving the use of life-saving hematopoietic transplants.

The human placenta is a hematopoietic organ during the embryonic and fetal periods of development.

We studied the potential role of the human placenta as a hematopoietic organ during embryonic and fetal development. Placental samples contained two cell populations-CD34(++)CD45(low) and CD34(+)CD45(low)-that were found in chorionic villi and in the chorioamniotic membrane. CD34(++)CD45(low) cells express many cell surface antigens found on multipotent primitive hematopoietic progenitors and hematopoietic stem cells. CD34(++)CD45(low) cells contained colony-forming units culture (CFU-C) with myeloid and erythroid potential in clonogenic in vitro assays, and they generated CD56(+) natural killer cells and CD19(+)CD20(+)sIgM(+) B cells in polyclonal liquid cultures. CD34(+)CD45(low) cells mostly comprised erythroid- and myeloid-committed progenitors, while CD34(-) cells lacked CFU-C. The placenta-derived precursors were fetal in origin, as demonstrated by FISH using repeat-sequence chromosome-specific probes for X and Y. The number of CD34(++)CD45(low) cells increased with gestational age, but their density (cells per gram of tissue) peaked at 5-8 wk, decreasing more than sevenfold at the onset of the fetal phase (9 wk of gestation). In addition to multipotent progenitors, the placenta contained myeloid- and erythroid-committed progenitors indicative of active in situ hematopoiesis. These data suggest that the human placenta is an important hematopoietic organ, raising the possibility of banking placental hematopoietic stem cells along with cord blood for transplantation.

Potential of Membranes Surrounding the Fetus as Immunoprotective Cell-Carriers for Allogeneic Transplantations.

BACKGROUND: Membranes surrounding the fetus play a crucial role in providing a physical and immunological barrier between a semiallogeneic fetus and mother during pregnancy. In this study, we tested whether cotransplantation of fetal membranes (FMs) and allogeneic donor cells would improve the retention and function of allografts in mice. METHODS: Intact and enzyme-digested membranes obtained from E18-E19 pregnant mice were subcutaneously cotransplanted with 10F7MN hybridoma cells that are of BALB/cByJ (Balb) origin and secrete anti-human CD235a antibody. Cells were transplanted into C57BL/6J (B6, allogeneic), Balb (syngeneic), and FVB/NJ (third-party) mice. Serum was collected after 1 and 3 weeks of cell transplantation and tested using flow cytometry for the presence of anti-human CD235a antibody. Immunosuppressive functions of membranes were further investigated by analyzing the cytokine profile of supernatants collected from allo-reactive mixed lymphocyte reactions (MLRs) using a multiplex cytokine assay. RESULTS: B6 mice transplanted with 10F7MN cells along with membranes syngeneic to the host had significantly higher levels of CD235a antibody when compared to B6 mice that received cells without membranes, allogenic membranes, or third-party membranes. Syngeneic membranes significantly inhibited T-cell proliferation in the presence of allogeneic stimuli and suppressed the release of Th1-cytokines such as IFNγ, TNFα, and IL-2 in MLRs. Additionally, increases in the levels of Th2-cytokines were found in MLRs containing membrane-derived cells. CONCLUSIONS: Our study highlights the potential use of syngeneic FMs to act as potent cell-carriers that could improve graft retention as well as graft-specific immunoprotection during allograft transplantation.

Maintenance of proliferative capacity and retroviral transduction efficiency of human fetal CD38(-)/CD34(++) stem cells.

Methods for the efficient transduction and expansion of fetal hematopoietic stem cells could lead to novel in utero therapies for blood cell disorders and enzymatic deficiencies. Here we describe a new assay to measure rapidly the effects of cytokines on the differentiation or expansion of primitive progenitors and stem cells found among CD38(-)CD34(++) lineage() cells isolated from human midgestation liver. Importantly, conditions that otherwise supported the expansion of clonogenic progenitors reduced their proliferative capacity. A combination of megakaryocyte growth and development factor and granulocyte-macrophage colony-stimulating factor maintained proliferative potential while also yielding an intermediate level of progenitor expansion. Retroviral transduction was achieved using Moloney murine leukemia virus-based vectors. Freshly isolated candidate stem cells could be transduced at almost 17% efficiency by a 1-h exposure to virus with centrifugation to aid transduction. This was increased to a mean 35.5% transduction efficiency after 1 day of culture. Additionally, the transduction efficiency of candidate stem cells isolated from fetal placental blood was 33.0%. These findings encourage further investigation into the feasibility of ex utero gene therapy whereby fetal cells are isolated from the circulation, transduced, and expanded ex utero before being returned to the fetus.

Preeclampsia and Inflammatory Preterm Labor Alter the Human Placental Hematopoietic Niche.

BACKGROUND: The human placenta is a source of hematopoietic stem and progenitor cells (HSPCs). The RUNX1 transcription factor is required for the formation of functional HSPCs. The impact of preeclampsia (PE) and preterm labor (PTL, spontaneous preterm labor [sPTL] and inflammatory preterm labor [iPTL]) on HSPC localization and RUNX1 expression in the human placenta is unknown. METHODS: We compared the frequency and density of HSPC in control samples from sPTL (n = 6) versus PE (n = 6) and iPTL (n = 6). We examined RUNX1 protein and RNA expression in placentas from normal pregnancies (5-22 weeks, n = 8 total) and in placentas from the aforementioned pregnancy complications (n = 5/group). RESULTS: Hematopoietic stem and progenitor cells were rare cell types, associated predominantly with the vasculature of placental villi. The HSPC density was greater in the chorionic plate (CP) compared to the villi (P < .001) and greater in PE and iPTL samples as compared to controls within the CP (not significant) and overall (P < .05). During the fetal period, RUNX1 was expressed in the mesenchyme of the CP and villi. Inflammatory PTL samples were more likely to exhibit intraluminal RUNX1(+) cell populations (P < .001) and RUNX1(+) cell clusters attached to arterial endothelial cells. CONCLUSION: Placental HSPCs likely arise from hematopoietic niches comprised RUNX1(+) mesenchyme and vascular endothelium. Pregnancy complications that result in preterm birth differentially affect placental HSPC localization and RUNX1 expression. Our results support previous findings that inflammation positively regulates hematopoiesis. We present new evidence that hemogenic endothelium may be active at later stages of human fetal development in the context of inflammation.

Identification of small activating RNAs that enhance endogenous OCT4 expression in human mesenchymal stem cells.

Ectopic overexpression of transcription factors has been used to reprogram cell fate. For example, virus-mediated overexpression of four transcription factors OCT4, SOX2, MYC, and KLF4, known as Yamanaka factors, can convert somatic cells to induced pluripotent stem (iPS) cells. However, gene-specific switch-on of endogenous gene production without the use of foreign DNA remains a challenge. The small RNA machinery that comprised small RNAs and Argonaute proteins is known to silence gene expression, but can be repurposed to activate gene expression when directed to gene promoters, a phenomenon known as RNA activation or RNAa. By screening of dsRNAs targeting OCT4 promoter, we identified a small activating RNA (saRNA) that activated OCT4 expression in several types of human mesenchymal stem cells (MSCs). We found that saRNA-induced OCT4 activation can be further enhanced by a histone deacetylase inhibitor, valproic acid. Furthermore, introducing OCT4 saRNA in combination with viruses encoding the remaining three Yamanaka factors (SOX2, MYC, and KLF4) into MSCs led to the derivation of partially reprogrammed iPS cells. Findings from this study suggest that, with further optimization, RNAa can be a powerful tool to reprogram cell fate by inducing the expression of endogenous genes.

Isolation, growth and identification of colony-forming cells with erythroid, myeloid, dendritic cell and NK-cell potential from human fetal liver.

The study of hematopoietic stem cells (HSCs) and the process by which they differentiate into committed progenitors has been hampered by the lack of in vitro clonal assays that can support erythroid, myeloid and lymphoid differentiation. We describe a method for the isolation from human fetal liver of highly purified candidate HSCs and progenitors based on the phenotypes CD38(-)CD34(++) and CD38(+)CD34(++), respectively. We also describe a method for the growth of colony-forming cells (CFCs) from these cell populations, under defined culture conditions, that supports the differentiation of erythroid, CD14/CD15(+) myeloid, CD1a(+) dendritic cell and CD56(+) NK cell lineages. Flow cytometric analyses of individual colonies demonstrate that CFCs with erythroid, myeloid and lymphoid potential are distributed among both the CD38(-) and CD38(+) populations of CD34(++) progenitors.

Ontogenic changes in CD95 expression on human leukocytes: prevalence of T-cells expressing activation markers and identification of CD95-CD45RO+ T-cells in the fetus.

The ontogeny of the human immune system was studied by analyzing fetal and adult tissues for the presence of various lymphocyte populations and activation/maturation markers. CD95 (fas) was expressed in hematopoietic tissues during the final stages of development of monocytes, granulocytes, NK cells and T cells, but to a much lesser extent on B cells. In the periphery, CD95 expression declined on granulocytes and NK cells. CD95 was expressed at a higher level on CD45RA+ peripheral T-cells in the fetus than in the adult. Contrary to the belief that most fetal T-cells are naïve or resting, a notable number of CD45RO+ T-cells were observed as well as an unique CD95-CD45RO+ population. Activation markers CD25, CD122, CD69 and CD80 were also present on fetal T-cells. These findings indicate that in the initial weeks following thymic maturation, a high frequency of T-cells is activated in the periphery of the fetus.

Stem cell transplantation in the fetus.

BACKGROUND: In utero transplantation (IUT) of hematopoietic stem cells has the potential to treat a large number of hematologic and metabolic diseases amenable to partial replacement of the hematopoietic system. METHODS: A review of the literature was conducted that focused on the clinical and experimental experience with IUT and, in this context, the development of the hematopoietic and immune systems. RESULTS: Successful application of IUT has been limited to the treatment of various types of immunodeficiencies that affect lymphocyte development and function. Other congenital defects such as the thalassemias have not resulted in clinically significant engraftment. Recent efforts at understanding and overcoming the barriers to engraftment in the fetus have focused on providing a selective advantage to donor stem cells and fostering immune tolerance toward the donor cells. The critical cellular components of the graft that promote engraftment and tolerance induction are being evaluated in animal models. Improvements in engraftment have resulted from the inclusion of T cells and/or dendritic cells in the graft, as well as a strategy of combined prenatal and postnatal transplantation. CONCLUSIONS: The advantages, necessity, and benefits of early treatment will continue to encourage development of IUT as a means to treat hematopoietic and other types of birth defects.

The adult livers of immunodeficient mice support human hematopoiesis: evidence for a hepatic mast cell population that develops early in human ontogeny.

The liver plays a vital role in hematopoiesis during mammalian prenatal development but its hematopoietic output declines during the perinatal period. Nonetheless, hepatic hematopoiesis is believed to persist into adulthood. We sought to model human adult-liver hematopoiesis by transplantation of fetal and neonatal hematopoietic stem cells (HSCs) into adult immunodeficient mice. Livers were found to be engrafted with human cells consisting primarily of monocytes and B-cells with lesser contributions by erythrocytes, T-cells, NK-cells and mast-cells. A resident population of CD117(++)CD203c(+) mast cells was also documented in human midgestation liver, indicating that these cells comprise part of the liver's resident immune cell repertoire throughout human ontogeny. The murine liver was shown to support human multilineage hematopoiesis up to 321 days after transplant. Evidence of murine hepatic hematopoiesis was also found in common mouse strains as old as 2 years. Human HSC engraftment of the murine liver was demonstrated by detection of high proliferative-potential colony-forming cells in clonal cultures, observation of CD38-CD34(++) and CD133(+)CD34(++) cells by flow cytometry, and hematopoietic reconstitution of secondary transplant recipients of chimeric liver cells. Additionally, chimeric mice with both hematopoietic and endothelial reconstitution were generated by intrasplenic injection of immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA) transgene. In conclusion, the murine liver is shown to be a hematopoietic organ throughout adult life that can also support human hematopoiesis in severely immunodeficient strains. Further humanization of the murine liver can be achieved in mice harboring an uPA transgene, which support engraftment of non-hematopoietic cells types. Thus, offering a model system to study the interaction of diverse human liver cell types that regulate hematopoiesis and immune function in the liver.

Coexpression of CD14 and CD326 discriminate hepatic precursors in the human fetal liver.

The molecular and cellular profile of liver cells during early human development is incomplete, complicating the isolation and study of hepatocytes, cholangiocytes, and hepatic stem cells from the complex amalgam of hepatic and hematopoietic cells, that is, the fetal liver. Epithelial cell adhesion molecule, CD326, has emerged as a marker of hepatic stem cells, and lipopolysaccharide receptor CD14 is known to be expressed on adult hepatocytes. Using flow cytometry, we studied the breadth of CD326 and CD14 expression in midgestation liver. Both CD45(+) hematopoietic and CD45(-) nonhematopoietic cells expressed CD326. Moreover, diverse cell types expressing CD326 were revealed among CD45(-) cells by costaining for CD14. Fluorescence-activated cell sorting was used to isolate nonhematopoietic cells distinguished by expression of high levels of CD326 and low CD14 (CD326(++)CD14(lo)), which were characterized for gene expression associated with liver development. CD326(++)CD14(lo) cells expressed the genes albumin, α-fetoprotein, hepatic nuclear factor 3α, prospero-related homeobox 1, cytochrome P450 3A7, α(1)-antitrypsin, and transferrin. Proteins expressed included cell-surface CD24, CD26, CD29, CD34, CD49f, CD243, and CD324 and, in the cytoplasm, cytokeratins-7/8 (CAM 5.2 antigen) and some cytokeratin-19. Cultured CD326(++)CD14(lo) cells yielded albumin(+) hepatocytes, cytokeratin-19(+) cholangiocytes, and hepatoblasts expressing both markers. Using epifluorescence microscopy we observed CD326 and CD14 expression on fetal hepatocytes comprising the liver parenchyma, as well as on cells associated with ductal plates and surrounding large vessels. These findings indicate that expression of CD14 and CD326 can be used to identify functionally distinct subsets of fetal liver cells, including CD326(++)CD14(lo) cells, representing a mixture of parenchymal cells, cholangiocytes, and hepatoblasts.

Detection of human hematopoietic stem cell engraftment in the livers of adult immunodeficient mice by an optimized flow cytometric method.

Immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mice are a valuable resource to study human hematopoietic stem cells. Prolonged multilineage hematopoiesis indicates stem cell engraftment and generally is measured by flow cytometry. In this study, we took advantage of the multi-parameter detection afforded by modern flow cytometers to optimize detection of human hematopoiesis in NSG mice. Antigens widely expressed by mouse or human cells were evaluated as markers to distinguish mixtures of these cells to optimize and test the limits of chimerism detection. The bone marrow, spleen, and liver of NSG mice transplanted with human hematopoietic cells were analyzed for evidence of engraftment.Mouse bone marrow cells were best marked for exclusion by staining with a combination of CD45, TER-119, and anti-H-2K(d) monoclonal antibodies, whereas live human cells were most accurately identified by elimination of cell doublets and positive staining for CD59. Human stem cells (CD34(++)CD133(+)CD38(low)) and progenitors were detected in the bone marrow and liver, but not in the spleen. An unusual pattern of myeloid antigen expression was detected in the bone marrow and CD3(+)CD4(+)CD8(+) T-cells were detected in the spleen. We concluded that multicolor flow cytometric analysis that clearly distinguishes mouse and human cells offers accurate detection of human chimerism in NSG mice. Human hematopoiesis can be detected in the bone marrow and liver of NSG mice with T-lymphopoiesis, possibly occurring in the spleen.

A new role for the human placenta as a hematopoietic site throughout gestation.

We investigated whether the human placenta contributes to embryonic and fetal hematopoietic development. Two cell populations--CD34(++)CD45(low) and CD34( +)CD45(low)--were found in chorionic villi. CD34(++) CD45(low) cells display many markers that are characteristic of multipotent primitive hematopoietic progenitors and hematopoietic stem cells. Clonogenic in vitro assays showed that CD34(++)CD45( low) cells contained colony-forming units-culture with myeloid and erythroid potential and differentiated into CD56(+) natural killer cells and CD19(+) B cells in culture. CD34(+)CD45(low) cells were mostly enriched in erythroid- and myeloid-committed progenitors. While the number of CD34(++)CD45(low) cells increased throughout gestation in parallel with placental mass. However, their density (cells per gram of tissue) reached its peak at 5 to 8 weeks, decreasing more than 7-fold from the ninth week onward. In addition to multipotent progenitors, the placenta contained intermediate progenitors, indicative of active hematopoiesis. Together, these data suggest that the human placenta is potentially an important hematopoietic organ, opening the possibility of banking placental hematopoietic stem cells along with cord blood for transplantation.

Megakaryocyte growth and development factor is a potent growth factor for primitive hematopoietic progenitors in the human fetus.

Megakaryocyte growth and development factor (MGDF), or thrombopoietin, has received considerable attention as a therapeutic agent for treating thrombocytopenia or for its use in the ex vivo culture of hematopoietic stem cells. MGDF is known to support the growth of a broad spectrum of hematopoietic precursors obtained from adult or neonatal tissues, but its effects on the growth of fetal progenitors and stem cells has not been studied. Human CD38(+)CD34(2+) progenitors and CD38(-)CD34(2+) cells, a population that contains stem cells, were isolated from midgestation liver and grown under defined conditions with MGDF and various cytokines known to support the growth of primitive hematopoietic precursors. In clonal assays of colony-forming cells (CFCs), MGDF supported the growth of 15-25% of candidate stem cells when combined with granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), flk-2/flt3 ligand, or stem cell factor. MGDF was observed to strongly support the early stages of hematopoiesis and expansion of high proliferative potential CFCs. More mature progenitors were expanded nearly 78-fold in 1 wk of culture with MGDF+SCF+GM-CSF. MGDF alone was also found to support the short-term (2 d) survival of CD38(-)CD34(2+) high proliferative potential CFCs. The effects of MGDF were more modest on CD38(+)CD34(2+) progenitors with only additive increases in colony formation being observed. These findings suggest that MGDF administration in fetuses and neonates may strongly affect the growth and mobilization of primitive hematopoietic progenitors and that MGDF may find use in the ex vivo growth and expansion of fetal stem cells.

Adeno-associated viral vector delivers cardiac-specific and hypoxia-inducible VEGF expression in ischemic mouse hearts.

It has been shown that the adeno-associated virus (AAV) vector can deliver the VEGF gene efficiently into the ischemic mouse myocardium. However, the AAV genomes can be found in extracardiac organs after intramyocardial injection. To limit unwanted VEGF expression in organs other than the heart, we tested the use of the cardiac myosin light chain 2v (MLC-2v) promoter and the hypoxia-response element to mediate cardiac-specific and hypoxia-inducible VEGF expression. An AAV vector, MLCVEGF, with 250 bp of the MLC-2v promoter and nine copies of the hypoxia-response element driving VEGF expression, was constructed. Gene expression was studied in vitro by infection of rat cardiomyocytes, rat skeletal myocytes, and mouse fibroblasts with the vector and in vivo by direct injection of the vector into normal and ischemic mouse hearts. With MLCVEGF infection, VEGF expression was higher in cardiomyocytes than the other two cell lines and was hypoxiainducible. VEGF expression was also higher in ischemic hearts than in normal hearts. No VEGF expression was detectable in organs with detectable MLCVEGF vectors other than the heart. MLCVEGF-injected ischemic hearts had more capillaries and small vessels around the injection site, smaller infarct size, and better cardiac function than the negative controls. Hence, MLCVEGF can mediate cardiac-specific and hypoxia-inducible VEGF expression, neoangiogenesis, infarct-size reduction, and cardiac functional improvement.