Clonal analysis of thymus-repopulating cells presents direct evidence for self-renewal division of human hematopoietic stem cells

To elucidate the in vivo kinetics of human hematopoietic stem cells (HSCs), CD34+CD38- cells were infected with lentivirus vector and transplanted into immunodeficient mice. We analyzed the multilineage differentiation and self-renewal abilities of individual thymus-repopulating clones in primary recipients, and their descending clones in paired secondary recipients, by tracing lentivirus gene integration sites in each lymphomyeloid progeny using a linear amplification-mediated polymerase chain reaction (PCR) strategy. Our clonal analysis revealed that a single human thymus-repopulating cell had the ability to produce lymphoid and myeloid lineage cells in the primary recipient and each secondary recipient, indicating that individual human HSCs expand clonally by self-renewal division. Furthermore, we found that the proportion of HSC clones present in the CD34+ cell population decreased as HSCs replicated during extensive repopulation and also as the differentiation capacity of the HSC clones became limited. This indicates the restriction of the ability of individual HSCs despite the expansion of total HSC population. We also demonstrated that the extensive self-renewal potential was confined in the relatively small proportion of HSC clones. We conclude that our clonal tracking studies clearly demonstrated that heterogeneity in the self-renewal capacity of HSC clones underlies the differences in clonal longevity in the CD34+ stem cell pool.     

Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation

Hematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy, but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular, the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified angiopoietin-like proteins and IGF-binding protein 2 (IGFBP2) as new hormones that, together with other factors, can expand mouse bone marrow HSCs in culture. Here, we measure the activity of multipotent human severe combined immunodeficient (SCID)-repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum-free medium containing SCF, TPO, and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133+ cells. Addition of either angiopoietin-like 5 or IGF-binding protein 2 to the cultures led to a sizable expansion of HSC numbers, as assayed by NOD/SCID transplantation. A serum-free culture containing SCF, TPO, FGF-1, angiopoietin-like 5, and IGFBP2 supports an approximately 20-fold net expansion of repopulating human cord blood HSCs, a number potentially applicable to several clinical processes including HSC transplantation.     

Unaltered repopulation properties of mouse hematopoietic stem cells transduced with lentiviral vectors

Recent studies of retroviral-mediated gene transfer have shown that retroviral integrations themselves may trigger nonmalignant clonal expansion of hematopoietic stem cells (HSCs) in transplant recipients. These observations suggested that previous conclusions of HSC dynamics based on gamma-retroviral gene marking should be confirmed with improved vectors having a more limited capacity to transactivate endogenous genes. Because of the low trans-activation activity of self-inactivating lentiviral vectors (LVs), we have investigated whether the LV marking of mouse HSCs induces a competitive repopulation advantage in recipients of serially transplants. As deduced from analyses conducted in primary and secondary recipients, we concluded that lentivirally transduced HSCs have no competitive repopulation advantages over untransduced HSCs. By linear amplification-mediated polymerase chain reaction (LAM-PCR) analysis, we characterized LV-targeted genes in HSC clones that engrafted up to quaternary recipients. Although 9 clones harbored integrations close to defined retroviral insertion sites, none was characterized as a common integration site, and none was present in HSC clones repopulating quaternary recipients. Taken together, our results show unaltered repopulation properties of HSCs transduced with LVs, and confirm early studies suggesting the natural capacity of a few HSC clones to generate a monoclonal or oligoclonal hematopoiesis in transplant recipients.     

The simple truth is seldom true and never simple: dual role for p75(NTR) in transdifferentation and cell death of hepatic stellate cells

Differentiation of hepatic stellate cells (HSCs) to extracellular matrix- and growth factor-producing cells supports liver regeneration through promotion of hepatocyte proliferation. We show that the neurotrophin receptor p75(NTR), a tumor necrosis factor receptor superfamily member expressed in HSCs after fibrotic and cirrhotic liver injury in humans, is a regulator of liver repair. In mice, depletion of p75(NTR) exacerbated liver pathology and inhibited hepatocyte proliferation in vivo. p75 (NTR-/-) HSCs failed to differentiate to myofibroblasts and did not support hepatocyte proliferation. Moreover, inhibition of p75(NTR) signaling to the small guanosine triphosphatase Rho resulted in impaired HSC differentiation. Our results identify signaling from p75(NTR) to Rho as a mechanism for the regulation of HSC differentiation to regeneration-promoting cells that support hepatocyte proliferation in the diseased liver.     

Osteoblastic expansion induced by parathyroid hormone receptor signaling in murine osteocytes is not sufficient to increase hematopoietic stem cells

Microenvironmental expansion of hematopoietic stem cells (HSCs) is induced by treatment with parathyroid hormone (PTH) or activation of the PTH receptor (PTH1R) in osteoblastic cells; however, the osteoblastic subset mediating this action of PTH is unknown. Osteocytes are terminally differentiated osteoblasts embedded in mineralized bone matrix but are connected with the BM. Activation of PTH1R in osteocytes increases osteoblastic number and bone mass. To establish whether osteocyte-mediated PTH1R signaling expands HSCs, we studied mice expressing a constitutively active PTH1R in osteocytes (TG mice). Osteoblasts, osteoclasts, and trabecular bone were increased in TG mice without changes in BM phenotypic HSCs or HSC function. TG mice had progressively increased trabecular bone but decreased HSC function. In severely affected TG mice, phenotypic HSCs were decreased in the BM but increased in the spleen. TG osteocytes had no increase in signals associated with microenvironmental HSC support, and the spindle-shaped osteoblastic cells that increased with PTH treatment were not present in TG bones. These findings demonstrate that activation of PTH1R signaling in osteocytes does not expand BM HSCs, which are instead decreased in TG mice. Therefore, osteocytes do not mediate the HSC expansion induced by PTH1R signaling. Further, osteoblastic expansion is not sufficient to increase HSCs.     

An intrinsic BM hematopoietic niche occupancy defect of HSC in scid mice facilitates exogenous HSC engraftment

Although scid mice have been widely used for human HSC engraftment studies, the function of HSCs of scid mice has not been characterized. We hypothesized that the DNA repair defect of scid mice results in a stem cell defect that facilitates HSC engraftment. scid BM cells showed severely impaired repopulation potentials in the competitive repopulation assay. To assess the BM hematopoietic niche occupancy ability of scid HSC, WT BM cells were transplanted into scid mice without any conditioning and observed to achieve long-term engraftment. Furthermore, the defects of scid HSCs are independent of their inability to perform lymphopoiesis because a similar defect in hematopoietic niche occupancy was not observed with Rag1(-/-) recipients. These results demonstrate that scid HSCs are impaired in maintenance within the niche, which may explain the nature of the conducive marrow niche environment of scid mice for xenotransplantation.     

Hematopoietic stem cell and progenitor defects in Sca-1/Ly-6A-null mice

Despite its wide use as a marker for hematopoietic stem cells (HSCs), the function of stem cell antigen-1 (Sca-1) (also known as lymphocyte activation protein-6A [Ly-6A]) in hematopoiesis remains poorly defined. We have previously established that Sca-1(-/-) T cells develop normally, although they are hyperresponsive to antigen. Here, we report detailed analysis of hematopoiesis in Sca-1-deficient animals. The differentiation potential of Sca-1-null bone marrow was determined from examination of the most mature precursors (culture colony-forming units [CFU-Cs]) to less committed progenitors (spleen CFUs [CFU-Ss]) to long-term repopulating HSCs. Sca-1-null mice are mildly thrombocytopenic with a concomitant decrease in megakaryocytes and their precursors. Bone marrow cells derived from Sca-1(-/-) mice also have decreased multipotential granulocyte, erythroid, macrophage, and megakaryocyte CFU (GEMM-CFU) and CFU-S progenitor activity. Competitive repopulation assays demonstrated that Sca-1(-/-) HSCs are at a competitive disadvantage compared with wild-type HSCs. To further analyze the potential of Sca-1(-/-) HSCs, serial transplantations were performed. While secondary repopulations using wild-type bone marrow completely repopulated Sca-1(-/-) mice, Sca-1(-/-) bone marrow failed to rescue one third of lethally irradiated wild-type mice receiving secondary bone marrow transplants from irradiation-induced anemia and contributed poorly to the surviving transplant recipients. These data strongly suggest that Sca-1 is required for regulating HSC self-renewal and the development of committed progenitor cells, megakaryocytes, and platelets. Thus, our studies conclusively demonstrate that Sca-1, in addition to being a marker of HSCs, regulates the developmental program of HSCs and specific progenitor populations.     

In vivo proliferation advantage of genetically corrected hematopoietic stem cells in a mouse model of Fanconi anemia FA-D1

Fanconi anemia (FA) is an inherited recessive DNA repair disorder mainly characterized by bone marrow failure and cancer predisposition. Studies in mosaic FA patients have shown that reversion of one inherited germ-line mutation resulting in a functional allele in one or a few hematopoietic stem cells (HSCs) can lead to the proliferation advantage of corrected cells, thus over time normalizing the hematologic status of the patient. In contrast to these observations, it is still unclear whether ex vivo genetic correction of FA HSCs also provides a similar proliferation advantage to FA HSCs. Using an FA mouse model with a marked hematopoietic phenotype, the FA-D1 (Brca2(Delta27/Delta27)) mice, we demonstrate that the lentivirus-mediated gene therapy of FA HSCs results in the progressive expansion of genetically corrected clones in mild-conditioned FA-D1 recipients. Consistent with these data, hematopoietic progenitors from FA recipients progressively became mitomycin C resistant and their chromosomal instability was reverted. No evidence of myelodysplasia, leukemias, or abnormal clonal repopulation was observed at multiple time points in primary or secondary recipients. Our results demonstrate that ectopic expression of BRCA2 confers a beneficial in vivo proliferation advantage to FA-D1 HSCs that enables the full hematopoietic repopulation of FA recipients with genetically corrected cells.     

Insulin-like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells

Hematopoietic stem cells (HSCs) undergo dramatic expansion during fetal liver development, but attempts to expand their numbers ex vivo have failed. We hypothesized that unidentified fetal liver cells produce growth factors that support HSC proliferation. Here we describe a novel population of CD3+ and Ter119- day-15 fetal liver cells that support HSC expansion in culture, as determined by limiting dilution mouse reconstitution analyses. DNA array experiments showed that, among other proteins, insulin-like growth factor 2 (IGF-2) is specifically expressed in fetal liver CD3+ cells but not in several cells that do not support HSCs. Treatment of fetal liver CD3+Ter119- cells with anti-IGF-2 abrogated their HSC supportive activity, suggesting that IGF-2 is the key molecule produced by these cells that stimulates HSC expansion. All mouse fetal liver and adult bone marrow HSCs express receptors for IGF-2. Indeed, when combined with other growth factors, IGF-2 supports a 2-fold expansion of day-15 fetal liver Lin-Sca-1+c-Kit+ long-term (LT)-HSC numbers. Thus, fetal liver CD3+Ter119- cells are a novel stromal population that is capable of supporting HSC expansion, and IGF-2, produced by these cells, is an important growth factor for fetal liver and, as we show, adult bone marrow HSCs.     

Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion

Ex vivo expansion of hematopoietic stem cells (HSCs) is important for many clinical applications, and knowledge of the surface phenotype of ex vivo-expanded HSCs will be critical to their purification and analysis. Here, we developed a simple culture system for bone marrow (BM) HSCs using low levels of stem cell factor (SCF), thrombopoietin (TPO), insulin-like growth factor 2 (IGF-2), and fibroblast growth factor-1 (FGF-1) in serum-free medium. As measured by competitive repopulation analyses, there was a more than 20-fold increase in numbers of long-term (LT)-HSCs after a 10-day culture of total BM cells. Culture of BM "side population" (SP) cells, a highly enriched stem cell population, for 10 days resulted in an approximate 8-fold expansion of repopulating HSCs. Similar to freshly isolated HSCs, repopulating HSCs after culture were positive for the stem cell markers Sca-1, Kit, and CD31 and receptors for IGF-2. Surprisingly, prion protein and Tie-2, which are present on freshly isolated HSCs, were not on cultured HSCs. Two other HSC markers, Endoglin and Mpl, were expressed only on a portion of cultured HSCs. Therefore, the surface phenotype of ex vivo-expanded HSCs is different from that of freshly isolated HSCs, but this plasticity of surface phenotype does not significantly alter their repopulation capability.     

SHIP deficiency enhances HSC proliferation and survival but compromises homing and repopulation

The SH2 domain-containing inositol 5'-phosphatase-1 (SHIP) has the potential to modulate multiple signaling pathways downstream of receptors that impact hematopoietic stem cell (HSC) biology. Therefore, we postulated that SHIP might play an important role in HSC homeostasis and function. Consistent with this hypothesis, HSC proliferation and numbers are increased in SHIP(-/-) mice. Despite expansion of the compartment, SHIP(-/-) HSCs exhibit reduced capacity for long-term repopulation. Interestingly, we observe that SHIP(-/-) stem/progenitor cells home inefficiently to bone marrow (BM), and consistent with this finding, have reduced surface levels of both CXCR4 and vascular cell adhesion marker-1 (VCAM-1). These studies demonstrate that SHIP is critical for normal HSC function, homeostasis, and homing.     

Hematopoietic senescence is postponed and hematopoietic stem cell function is enhanced by dietary restriction

OBJECTIVE: The aim of this study was to test dietary restriction (DR) as an intervention to alleviate senescence-associated functional defects in hematopoietic stem cells (HSCs). MATERIALS AND METHODS: BALB/cByJ (BALB) mice were fed ad libitum (AL) or were diet restricted (DR) to 75% of the AL food intake after 1 month of age. Peripheral blood and bone marrow cell compositions were compared in 3- and 25-month-old AL (AL-3, AL-25) mice and in 25-month-old DR (DR-25) mice using fluorescence-activated cell staining. Relative HSC functions in vivo were compared using competitive repopulation, and were also tested in 6-month-old BALB mice to measure the effects of short-term DR. RESULTS: Compared to AL-3, AL-25 blood had significantly lower levels of red blood cells and hemoglobin. AL-25 marrow contained less than half the concentration of Lin(-)CD34(-)Sca1(+)CD117(+) HSCs and showed only half the in vivo functional ability of AL-3 marrow. In vivo, AL-25 HSCs failed to produce the strong correlations over time that demonstrate clonal stability during competitive repopulation. These correlations were shown in AL-3 HSCs. DR for 24 months alleviated hematopoietic deficiencies in the blood, increased concentrations of bone marrow Lin(-)CD34(-)Sca1(+)CD117(+) HSCs and improved HSC functional abilities in DR-25 mice to values far greater than those in normally aged mice. Surprisingly, HSC function in 25-month-old DR mice was better than that in young adults. Degrees of recipient repopulation by HSCs from DR-25 mice also correlated well over time, demonstrating clonal stability. Short-term DR for 5 months also improved HSC function, but to a much smaller degree. CONCLUSIONS: Aged BALB mice show hematopoietic and HSC senescence and clonal succession. Lifelong DR slows hematopoietic senescence and prevents HSC aging.     

Inhibition of p38 MAPK activity promotes ex vivo expansion of human cord blood hematopoietic stem cells

Ex vivo expansion of hematopoietic stem cells (HSCs) depends on HSC self-renewing proliferation and functional maintenance, which can be negatively affected by HSC differentiation, apoptosis, and senescence. Therefore, inhibition of HSC senescence may promote HSC expansion. To test this hypothesis, we examined the effect of inhibition of p38 mitogen-activated protein kinase (p38) on the expansion of human umbilical cord blood (hUCB) CD133⁺ cells because activation of p38 has been implicated in the induction of HSC senescence under various physiological and pathological conditions. Our results showed that ex vivo expansion of hUCB CD133⁺ cells activated p38, which was abrogated by the p38 specific inhibitor SB203580 (SB). Inhibition of p38 activity with SB promoted the expansion of CD133⁺ cells and CD133⁺CD38⁻ cells. In addition, hUCB CD133⁺ cells expanded in the presence of SB for 7 days showed about threefold increase in the clonogenic function of HSCs and engraftment in non-obese diabetic/severe combined immunodeficient mice after transplantation compared to the input cells. In contrast, the cells expanded without SB exhibited a significant reduction in these HSC functions. The enhancement of ex vivo expansion of hUCB HSCs is primarily attributable to SB-mediated inhibition of HSC senescence. In addition, inhibition of HSC apoptosis and upregulation of CXCR4 may also contribute to the enhancement. However, p38 inhibition had no significant effect on HSC differentiation and proliferation. These findings suggest that inhibition of p38 activation may represent a novel strategy to promote ex vivo expansion of hUCB HSCs.     

Critical role of tumor necrosis factor receptor 1, but not 2, in hepatic stellate cell proliferation, extracellular matrix remodeling, and liver fibrogenesis

Tumor necrosis factor (TNF) has been implicated in the progression of many chronic liver diseases leading to fibrosis; however, the role of TNF in fibrogenesis is controversial and the specific contribution of TNF receptors to hepatic stellate cell (HSC) activation remains to be established. Using HSCs from wild-type, TNF-receptor-1 (TNFR1) knockout, TNF-receptor-2 (TNFR2) knockout, or TNFR1/R2 double-knockout (TNFR-DKO) mice, we show that loss of both TNF receptors reduced procollagen-α1(I) expression, slowed down HSC proliferation, and impaired platelet-derived growth factor (PDGF)-induced promitogenic signaling in HSCs. TNFR-DKO HSCs exhibited decreased AKT phosphorylation and in vitro proliferation in response to PDGF. These effects were reproduced in TNFR1 knockout, but not TNFR2 knockout, HSCs. In addition, matrix metalloproteinase 9 (MMP-9) expression was dependent on TNF binding to TNFR1 in primary mouse HSCs. These results were validated in the human HSC cell line, LX2, using neutralizing antibodies against TNFR1 and TNFR2. Moreover, in vivo liver damage and fibrogenesis after bile-duct ligation were reduced in TNFR-DKO and TNFR1 knockout mice, compared to wild-type or TNFR2 knockout mice. CONCLUSION: TNF regulates HSC biology through its binding to TNFR1, which is required for HSC proliferation and MMP-9 expression. These data indicate a regulatory role for TNF in extracellular matrix remodeling and liver fibrosis, suggesting that targeting TNFR1 may be of benefit to attenuate liver fibrogenesis.     

Influence of telomere length on short-term recovery after allogeneic stem cell transplantation

OBJECTIVE: Telomeres shorten in somatic cells during aging and states of increased turnover, including hematopoietic stem cell transplantation. Fast hematopoietic recovery is critical for the patients' course after hematopoietic stem cell transplantation. It is unknown whether telomere length in hematopoietic stem cells (HSCs) predicts short-term hematopoietic recovery. METHODS: We quantified telomere length by flow fluorescence in situ hybridization analysis in HSCs and granulocytes of healthy stem cell donors and monitored time to peripheral blood cell recovery in transplanted hosts. Furthermore, we measured in vitro repopulation potency of HSCs by assaying for colony-forming units granulocyte-macrophage (CFU-GM). RESULTS: Telomere length in HSC shortens continuously in vivo and is comparable to telomere length in granulocytes from the same individual. Numbers of in vitro formed CFU-GM per HSC show an inverse relationship to age and telomere length. However, telomere length in HSCs was not correlated with short-term recovery after HSC transplantation. CONCLUSION: These findings suggest that healthy stem cell donors have sufficient telomere length reserve to repopulate a myeloablatively treated host, despite continuous aging of HSCs in vivo and decreased repopulation ability of HSCs from older donors in vitro.     

Long-term repopulation of hematolymphoid cells with only a few hemopoietic stem cells in mice.

A PCR-based assay has been devised for the detection and semiquantitation of cells originating from a few donor hematopoietic stem cells (HSCs) in a background of recipient cells. Upon sequencing a segment of murine Y chromosome contained in the plasmid pY2, oligonucleotide primers were designed for specific amplification of the Y chromosome-restricted segment. The HSCs were isolated from the bone marrow of mice on day 4 following a single i.v. injection of 5-fluorouracil and were readily distinguished from other bone marrow elements by the characteristics of low density, absence of lineage-specific surface markers, lack of expression of transferrin receptor, and a high expression of major histocompatibility complex class I antigen. Injection of as few as four such HSCs was shown to produce donor-derived cells (including lymphoid cells) for at least 8 months after transplantation into syngeneic female recipients. Retransplantation, employing 10(6) bone marrow cells from the initial recipients, also yielded clear evidence of repopulation with detectable levels of male donor cells. On statistical grounds, it is clear that long-term repopulation in vivo may result from even a single HSC having the characteristics defined herein.