Relative quiescence is a defining characteristic of hematopoietic stem cells, while their progeny have dramatic proliferative ability and inexorably move toward terminal differentiation. The quiescence of stem cells has been conjectured to be of critical biologic importance in protecting the stem cell compartment, which we directly assessed using mice engineered to be deficient in the G1 checkpoint regulator, cyclin-dependent kinase inhibitor, p21cip1/waf1 (p21). In the absence of p21, hematopoietic stem cell proliferation and absolute number were increased under normal homeostatic conditions. Exposing the animals to cell cycle-specific myelotoxic injury resulted in premature death due to hematopoietic cell depletion. Further, self-renewal of primitive cells was impaired in serially transplanted bone marrow from p21-/- mice, leading to hematopoietic failure. Therefore, p21 is the molecular switch governing the entry of stem cells into the cell cycle, and in its absence, increased cell cycling leads to stem cell exhaustion. Under conditions of stress, restricted cell cycling is crucial to prevent premature stem cell depletion and hematopoietic death.

The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, drug screening and modeling of lung disease, and studies of human lung development. We established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. Type II alveolar epithelial cells generated were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing agonists to signaling pathways critical for early lung development in the mouse—retinoic acid, Wnt and BMP—recapitulated defects in corresponding genetic mouse knockouts. The capability of this protocol to generate most cell types of the respiratory system suggests its utility for deriving patient-specific therapeutic cells.

IntroductionHuman hematopoietic tissue and cell transplantation into immunodeficient mice, such as mice with the severe combined immunodeficiency (SCID) 1,2 mutation and their derivatives, including NOD/SCID, 3 NOD/SCID/␤2m null , 4 and NOD/SCID/ ␥c null mice, 5 has been widely used to study the function of human immune cells. However, only a few studies have shown the ability to achieve systemic in vivo immune responses in humanized mice. 6 In general, immunodeficient mice that received a transplant of human hematopoietic stem cells (HSCs) have poor human T-cell development. Although some human T-cell reconstitution was seen in NOD/SCID/␥c null mice after human umbilical cord blood (UCB) stem-cell transplantation, human thymopoiesis in the recipient thymus appeared inefficient, and the mouse thymus remained underdeveloped in these mice. 5 Recently, Traggiai et al 6 reported that intrahepatic injection of human CD34 ϩ UCB cells into newborn Rag2 Ϫ/ Ϫ␥c Ϫ/Ϫ mice results in improved human T-cell development in the mouse thymus. An important advancement in this model compared with UCB transplantation in adult mice was the ability of grafted mice to mediate in vivo antiviral immune responses. 6 However, a long period of time (16 weeks) was required to achieve significant peripheral human T-cell repopulation in these mice, and the levels of human T cells in the spleen of long-term surviving mice were relatively low. Furthermore, the MHC restriction and tolerance status of human T cells that were selected in the mouse thymus in these mice still remains undefined.A commonly used mouse model for the study of human For personal use only. on June 19, 2019. by guest www.bloodjournal.org From Materials and methods Animals and human fetal tissuesImmunodeficient nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were housed in a specific pathogen-free microisolator environment and used at 6 to 10 weeks of age. Human fetal thymus and liver tissues of gestational age of 17 to 20 weeks were obtained from Advanced Bioscience Resource (Alameda, CA). Inbred Massachusetts General Hospital miniature swine (kindly provided by David H. Sachs) 10 were used as porcine skin donors. Protocols involving the use of human tissues and animals were approved by the Massachusetts General Hospital Human Research Committee and Subcommittee on Research Animal Care, and all of the experiments were performed in accordance with the protocols. Human tissue implantationMice were conditioned with sublethal (2-3 Gy) whole-body irradiation. Human fetal Thy and Liv fragments measuring about 1 mm 3 were implanted under the recipient kidney capsule within 3 days after irradiation. Some mice also received CD34 ϩ FLCs (1-5 ϫ 10 5 /mouse, intravenously) purified from the same donor on the day of human Thy/Liv transplantation. CD34 ϩ FLCs were isolated by the magnetic-activated cell sorter (MACS) separation system using anti-CD34 microbeads (Miltenyi Biotec, Auburn, CA). Levels of human hematopoietic cells in the mice that underwent transplantation ...