Tissue source dictates lineage outcome of human fetal CD34+CD38− cells

Experimental Hematology 29 (2001) 766–774

Tissue source dictates lineage outcome of human fetal CD34CD38 cells Mark C. Poznansky, Ivona T. Olszak, Russell B. Foxall, Anita Piascik, Gregor B. Adams, Richard H. Evans, Tao Cheng, and David T. Scadden AIDS Research Center and MGH Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, Mass., USA (Received 20 November 2000; revised 22 January 2001; accepted 25 January 2001)

Objective. The translocation from fetal liver hematopoiesis to secondary organs occurs during the second trimester of human gestation. It has been hypothesized that stem cells migrate and acquire lineage potential based on cues specific to the adopted microenvironment. We evaluated primitive hematopoietic cell populations in the fetal human to determine if lineage restriction precedes or follows translocation to sites of hematopoietic activity including thymus, spleen, bone marrow, and liver. Methods. Sets of hematopoietic tissues from individual second-trimester human abortuses were used to compare and quantitate the lineage outcome of immunophenotypically primitive cells from each of the hematopoietic organs using ex vivo myeloid and lymphoid differentiation systems. Results. Despite uniformity in immunophenotype, functional capabilities were highly restricted by the tissue of origin and alteration in the ex vivo differentiation context did not lead to a change in differentiation outcome. Conclusion. Translocation of primitive cells from fetal liver to tissues of mature hematopoietic activity is associated with tissue-specific, quantitative changes in differentiation potential that are unresponsive to alternative differentiation environments. These data suggest that multipotentiality is lost prior to or upon stem-cell migration in the developing human. It is not persistent with residence in a secondary hematopoietic organ. © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Organization of the hematopoietic system shifts during ontogeny, accomplishing specific tasks at particular times during development [1,2,3]. The largely erythrogenerative function of the primitive hematopoietic system is replaced by a more complex mix of myeloid cell production in the fetal liver [4]. During the second trimester, stem cell populations move from fetal liver to sites of continued broad myeloid production in the bone marrow and sites of more specialized lymphoid production in the thymus and spleen [4–7]. This period of migration to sites of hematopoietic activity of the mature organism represents a unique window for examining the functional potential of immunophenotypic subsets of primitive cells. Cells bearing lineage-specific markers define a population of cells that have moved irreversibly down specific lineage differentiation paths, but cells that are CD34 and negative for the differentiation marker, CD38, have been shown to represent a primitive Offprint requests to: David T. Scadden, M.D., Massachusetts General Hospital, 149 13th Street, Room 5212, Boston, MA 02129; E-mail: scadden. [email protected]

cell compartment with multipotentiality [8–11]. Recent studies suggest that there is a continuum of primitive cells from those truly pluripotent to multipotent cells. The data presented here may reflect only those cells at the more committed end of that spectrum of stem-cell types in that we restricted our analysis to the multipotent, CD34CD38 immunophenotype. This multipotential pool results in specific cell lineage descendents based on molecular events that have been variably argued to be dominated by either extrinsic cues from the microenvironment or intrinsic programs independent of exogenous signal type [12,13]. The timing of lineage commitment is not well defined, but is thought to occur as cells transit out of the CD34CD38 phenotype [8,9,11,14]. Similarly, the plasticity of commitment is not clear and, to the extent that microenvironment dictates instructive cues, may be preserved until specific differentiation markers appear after CD38 expression is detectable [9,11]. The availability of hematopoietic organ sets from individual abortuses, including bone marrow, thymus, spleen, liver, and blood, during the time of stem-cell translocation

0301-472X/01 $–see front matter. Copyright © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(01)0 0 6 4 3 - 9

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offers the opportunity to assess lineage commitment among immunophenotypically homogeneous CD34CD38 cells without donor-specific variability. Through the comparative quantitation of lymphoid vs myeloid outcomes from cells of different hematopoietic organs from the same individual, we documented that multipotentiality may be lost prior to or at the time of emigration.

Methods Fetal organ harvest and mononuclear cell preparation Human fetal bone marrow, liver, thymus, spleen, and peripheral blood were obtained from each of six human fetal abortuses (gestational ages 16–22 weeks) according to Institutional Review Board–approved guidelines. Mononuclear cell preparations were obtained from liver, thymus, and spleen following disaggregation of the organ with or without 0.5mg/mL collagenase (Sigma, St. Louis, MO, USA). Bone marrow mononuclear cells were harvested from fetal bone by perfusion of the fetal femur with Iscove’s medium. Mononuclear cells isolated from human fetal organs were layered over Histopaque (Sigma) and centrifuged at 2000 rpm for 20 minutes in order to remove contaminating red cells. Flow cytometric analysis of mononuclear cells and purified CD34 cell populations Mononuclear cells from bone marrow, liver, blood, thymus, and spleen were stained using fluorescein isothiocyanate (FITC)–labeled anti-CD2, CD3, CD4, CD8, CD13, CD19, CD33, CD34, CD38, CD45, CD45RA, CD45RO, CD56, and CD71 (BectonDickinson, San Jose, CA, USA). Cells positive for labeling with each or combinations of these fluorescent-labeled antibodies were identified and quantitated using a FACScalibur flow cytometer (Becton-Dickinson). CD34 cell purification, culture, and colony-forming cell (CFC) assays Mononuclear cells were cultured overnight in Iscove’s modified Dulbecco’s medium (IMDM; Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) at 37C/5% CO2 in a humidified atmosphere to remove the adherent cells prior to CD34 cell isolation using the MiniMACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified CD34 cells were then incubated with anti-CD2 immunomagnetic beads to deplete T-lineage committed cells. The purity of the CD34 cells prepared by this method was greater than 98% and contained undetectable levels of CD2 positivity by flow cytometry. Aliquots of purified CD34CD2 cells from each of the organs were then labeled with anti-CD38 (Becton-Dickinson) and CD34CD38 subpopulations were purified using the FACSvantage cell sorter. Quantitative assessment of myeloid/erythroid potential of CD34CD38 cells: CFC and LTC-IC assays Isolated CD34CD2 cells and sorted CD34CD38 cells that were derived from CD2-depleted CD34 cells were mixed with IMDM-based methylcellulose media (MethoCult GF H4434, Stem Cell Technologies, Vancouver, Canada) containing 30% fetal bovine serum, 1% bovine serum albumin, 3 U/mL erythropoietin,

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104 M 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/mL recombinant human stem cell factor, 10 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor, and 10 ng/mL recombinant human interleukin-3 to give a concentration of 2  103 cells/mL. Cells were plated in duplicate in 24-well tissue-culture plates and incubated for 2 weeks at 37C/5% CO2 in a humidified atmosphere, following which the cultures were scored for burstforming unit erythroid (BFU-E), colony-forming unit granulocytemacrophage (CFU-GM), or colony-forming unit granulocyteerythroid-macrophage-megakaryocyte (CFU-GEMM) according to standard phase microscopic criteria. The presence of long-term culture-initiating cells (LTC-IC) was assessed by limiting dilution of the cells on a primary human bone marrow stromal feeder layer for 5 weeks as previously described [15]. Briefly, low-density bone marrow cells were cultured at 37C for 3–4 days prior to transfer to 33C in long-term culture (LTC) medium (-MEM with 12.5% horse serum, 12.5% fetal bovine serum, 0.2 mM I-inositol, 20 mM folic acid, 104 M 2-mercaptoethanol, 2 mM L-glutamine (StemCell Technologies Inc., Vancouver, B.C., Canada) and 106 M hydrocortisone (Sigma)). The confluent stroma layer was trypsinized, irradiated (15 Gy), and subcultured in 96-well flat-bottomed plates at a density of 1.25  104/well. Within one week, sorted CD34CD38 cells from human fetal liver, bone marrow, blood, spleen, or thymus were seeded at 3–5 dilutions with 12 replicate wells per cell concentration and cultured at 33C for 5 weeks with half-volume medium changes weekly. Culture plates were then centrifuged. IMDM semisolid medium (StemCell Technologies) containing 0.9% methylcellulose, 30% fetal bovine serum, 1% bovine serum albumin, 104 M 2-mercaptoethanol, and 2 mM L-glutamine, supplemented with 20 ng/mL interleukin-3, 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 50 ng/mL stem cell factor (SCF; R & D Systems, Minneapolis, MN, USA), 20 ng/mL granulocyte colony-stimulating factor (G-CSF), and 3 U/mL erythropoietin (Amgen Inc., Thousand Oaks, CA, USA) was overlaid. Following 10 days at 37C, 5% CO2, colonies were quantitated by phase-contrast microscopy and the LTC-IC frequency was calculated using either Maxrob software or linear regression analysis. Quantitative assessment of T-lymphoid potential of CD34CD38 cells using a murine thymic organoid Murine thymus was obtained from 4–6-week-old C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME, USA). The thymus was disaggregated in IMDM containing 10% heat-inactivated fetal calf serum with sterile surgical scissors to a fragment size below 0.5 mm3 within a suspension of murine thymic mononuclear cells at a concentration of approximately 1  107 cells/mL. 0.5 mL of the mononuclear cell suspension containing between 3 and 5 thymic fragments was plated onto a three-dimensional matrix, CellFoam (0.5 cm  0.5 cm  0.2 cm with 80 pores per square inch) (Cytomatrix, Woburn, MA, USA), in each of the 24 wells of a 24-well tissue culture plate, and a further 0.5 mL of supplemented IMDM was then added to each well. The medium in the wells was changed at 48 hours post establishment of the culture and every 72 hours thereafter. Murine thymic stroma reached 70% confluency on the three-dimensional matrix at 10–14 days of culture and at this time the stroma was irradiated at 15 Gy. Each cell foam unit with near-confluent irradiated stroma was then removed from the original well in which it was cultured and placed in a new well in a 24-well plate in fully supplemented IMDM. The murine thymic

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stroma was then cultured for a further 24 hours prior to progenitor cell addition. Sorted CD34CD38 cells derived from CD34CD2 cells were cocultured at 5  103/well, 5  102/well or 50/well with irradiated murine thymic stroma containing cell foam units without exogenous cytokines for 14 days. Half of the medium in the cocultures was gently replaced with fresh supplemented medium every 72 hours. Two assays were used to quantitate the number, phenotype, and function of the cells generated in the coculture. First, the immunophenotype and number of cells generated in the murine thymic/ human fetal CD34 cell coculture system was analyzed at 7 and 14 days. Cells were stained with anti-CD3, CD4, CD8, CD14, CD33, and CD45 antibodies (Becton-Dickinson, San Jose, CA, USA). The immunophenotype of mononuclear cells harvested from murine thymic stroma alone was also analyzed for T-cell markers. The proportion of CD45 cells bearing CD3CD4, CD3CD8, CD4CD8, and CD14CD33 generated in the cultures was determined. Secondly, CD3CD4 and CD3CD8 generated from the coculture were subsequently stimulated with the mitogens PHA and Con A and proliferation quantitated in the presence of tritiated thymidine. Statistical analysis Numerical data was compared using the Student’s t-test.

Results Immunophenotype of the mononuclear fraction of human fetal hematopoietic organs The immunophenotype of cells from liver, spleen, thymus, bone marrow, and peripheral blood was examined by flow cytometry for expression of CD1, CD2, CD3, CD4, CD8,

CD13, CD19, CD33, CD45, CD45RA, CD45RO, CD56, and CD71 (Table 1 and Fig. 1). There was minimal variability between the immunophenotypic analysis of mononuclear cells from the same organs from different individuals with SEM values varying between 5.0% and 8.2%. However, there were distinct and significant differences with regard to the immunophenotype of mononuclear cell fractions between different organs in the same individual fetus. The mononuclear fractions of liver and bone marrow resembled each other with regard to mature lymphoid and myeloid cells and precursor cells. The exception was transferrin receptor (CD71), which was significantly higher in the fetal liver compared with fetal bone marrow, likely reflecting the erythrogenic predominance of fetal liver (p  0.001: Student’s t-test). Fetal liver and bone marrow had comparable levels of CD34 cells, but a significantly smaller proportion of CD34CD38 cells were noted in the fetal bone marrow compared with liver, suggesting a shift toward more mature populations of progenitors in the marrow space (Table 1 and Fig. 1). The immunophenotype of mononuclear cells derived from spleen and thymus clearly differed from liver and bone marrow and the immunophenotype of cells from thymus clearly differed from that of spleen. Spleen contained significantly higher levels of mature T-cell markers CD3, CD4, and CD8 cells than liver or bone marrow (p  0.01: Student’s t-test). Unexpectedly, spleen was not substantially different from liver or bone marrow with regard to cells bearing the B-lineage marker, CD19. Also unexpected, spleen contained a high proportion of CD34 cells with a predominance of mature CD34CD38 cells and an abundance of CD56 cells, suggesting a large natural killer (NK)

Table 1. Immunophenotype of mononuclear cells from human fetal hematopoietic organs: 16–22 weeks gestation (n  5) Percent of total MNC

Liver

Blood

Bone marrow

Spleen

Thymus

CD2 CD3 CD4 CD8 CD13 CD19 CD33 CD45 CD45RA CD45RO CD56 CD71 CD34 CD34CD38 (% of CD34 cells)

11.0 1.1% 9.0 1.0% 5.0 0.4% 8.0 0.9% 8.0 0.6% 39.0 3.1% 13.0 0.9% 65.0 2.1% 69.0 4.1% 9.0 0.6% 8.0 0.3% 71.0 0.7% 26.0 2.1%

47.0 2.4% 44.0 2.3% 33.0 1.8% 17.0 0.8% 2.0 0.1% 25.0 1.7% 3.0 0.2% 80.0 3.5% 75.0 3.2% 14.0 0.7% 12.0 0.8% 24.0 1.1% 5.0 0.2%

10.0 0.1% 7.0 0.3% 3.0 0.1% 2.0 0.2% 2.0 0.1% 33.0 2.3% 11.0 0.6% 98.0 1.0% 75.0 3.9% 6.0 0.2% 2.0 0.1% 7.0 0.3% 28.0 1.2%

26.0 1.5% 22.0 2.0% 17.0 1.2% 12.0 0.8% 2.0 0.1% 32.0 1.9% 3.0 0.4% 70.0 3.7% 76.0 4.3% 8.0 0.5% 15.0 0.1% 13.0 0.1% 28.0 0.2%

96.0 4.2% 74.0 2.6% 83.0 3.5% 62.0 2.7% 3.0 0.1% 3.0 0.1% 2.0 0.3% 95.0 2.3% 5.0 0.2% 86.0 2.8% 2.0 0.1% 2.0 0.1% 7.0 0.2%

49.0 4.9%

17.0 1.8%

25.0 2.3%

33.0 4.4%

8.0 0.9%

Mononuclear cell fractions were obtained from liver, blood, bone marrow, spleen, and thymus of each of 5 human fetuses (16–22 weeks gestation). Mononuclear cells were examined for expression of CD2, CD3, CD4, CD8, CD13, CD19, CD33, CD34, CD45, CD45RA, CD45RO, CD56, and CD71. The percentages of cells expressing these cell surface markers SEM are shown. Immunophenotype of CD34 cells from human fetal hematopoietic organs. CD34 cells were isolated from the mononuclear fractions of fetal liver, blood, bone marrow, spleen, and thymus of each of 5 human fetuses (16–22 weeks gestation). Mononuclear cells were examined for expression of CD34 and CD38. The percentages of cells expressing these cell surface markers ( SEM) are shown.

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Figure 1. The immunophenotype of mononuclear cells from human fetal hematopoietic organs. Staining for (A) CD19, CD3, CD33, or (B) CD34 and CD38 are shown for each of the hematopoietic organs from a single individual human fetal abortus.

population. Thymic mononuclear cells (MNC) expressed T-cell markers and a small proportion of CD34 cells, which were also predominantly CD34CD38. Fetal blood in the 16–22 week fetus demonstrated CD3 staining significantly greater than liver, bone marrow, and spleen (p  0.005: Student’s t-test) but significantly less than thymus (p  0.01: Student’s t-test), suggesting full maturation of T cells by the second trimester with egress from the thymus into the circulation. The proportion of CD34 cells in peripheral blood was the lowest for all hematopoietic organs studied (5.0 0.2%); however, the absolute number was likely to be high given the blood volume and reflects the active translocation of stem cells thought to occur during this gestational period.

Assessment of lineage potential of human fetal CD34CD38 cells Purified CD34CD38 cells were used to define the differentiation potential in distinct assays of T-lymphoid and myeloid differentiation. Aliquots of identical immunopheno-

typic cells were compared and their myeloid and T-lymphoid potential quantitated. We defined multipotential as the ability to revert to an alternative lineage fate, with input cells simultaneously assayed in both T-lymphoid and myeloid differentiation systems. Lineage yield we defined by the productive capacity of primitive cells within a given lineage assay, such as the CFC generated from a given input number of cells. T-lymphopoietic capacity of CD34CD38 cells from human fetal organs The T-lymphoid potential of CD34CD38 cells was assessed using a coculture with murine thymic stroma that had been established on a coated tantalum three-dimensional matrix. This system utilizes CD2-depleted progenitor cells that are RT-PCR negative for T-cell receptor excision circle (TREC) and generates single-positive T cells expressing a complex repertoire of T-cell receptor (TCR) V chains and a proportion of cells bearing TCR excision circles equivalent to that of the human fetal thymus [16]. In addition, the cells proliferated following stimulation with mitogens and

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interleukin-2 and are infectible with T-tropic strains of HIV-1. The assay has been adapted to provide low-variability, semiquantitative assessments of T-lymphopoietic capacity of input cells. An input of 5000 fetal thymus CD34CD38 cells generated the greatest number and proportion of CD45 cells bearing T-cell markers at two weeks including 58.1 3.9% CD3CD4 cells, 12.1 3.6% CD3CD8 cells, and 11.1 0.1% CD4CD8 cells (Table 2). In contrast, fetal liver, blood, and bone marrow CD34CD38 cells generated less than one-third that number (10.9–18.1% CD3CD4 cells, 0.5–4.3% CD3CD8 cells, and 0.8–2.1% CD4CD8 cells). Spleen CD34 and CD34CD38 cells did not proliferate or result in detectable CD45 human T-cell progeny (Table 2). Titering down the input cells to 500 fetal CD34CD38 cells was below the threshold for generating T cells for all tissues except the thymus. Even for thymusderived cells, further titration of input cells to 50 or fewer input progenitor cells resulted in no T-cell output (data not shown). These data demonstrate the limited T-lymphoid potential of primitive cells from liver (as previously shown by Barcena et al. in the human [17] and Kawamoto et al. in the mouse [18,19]), bone marrow, or blood and depletion of any T-lymphopoietic capacity among spleen-derived CD34CD38 cells. To the extent that the titration of input cells reflects T-lymphopoietic potential, thymic CD34CD38 cells appear to be superior to other tissue sources by approximately an order of magnitude. Both immature double-positive cells (CD4CD8) and single-positive (CD3CD4 and CD3CD8) T cells were generated by day 14 of the coculture of fetal CD34 CD38 cells from thymus, bone marrow, blood, and liver with murine thymic stroma (Fig. 2). CD3 T cells generated by day 14 in the coculture proliferated in response to T-cell mitogens (Fig. 2). Therefore, if cells were capable of undergoing

T-lymphoid differentiation the lineage yield was comparable in subtype and function, though different in number. Cells bearing the myelomonocytic markers, CD14 and CD33, were also generated in the coculture system and composed virtually all of the cells outside the lymphoid gate. Liver, bone marrow, and blood CD34CD38 cells generated significantly greater proportions and numbers of CD14CD33 cells than did thymic CD34CD38 cells on murine thymic stroma (p  0.0001: Student’ t-test) (Table 2), suggesting that the inability to respond to T-cell differentiation signals is due to preceding myeloid commitment. CFC and LTC-IC potential of CD34CD38 cells Having demonstrated the highly tissue-specific T-lymphopoietic capacity of CD34CD38 cells, we evaluated the same cells under conditions favoring myeloid differentiation to test if the cells retained full multipotentiality when placed in an alternative milieu. CFU-GM, BFU-E, CFUmix, and total CFC potential of CD34CD38 cells was assessed using standard methylcellulose assays. CFC frequency for CD34CD38 cells was significantly greater ( p  0.01: Student’s t-test) for liver than for all other hematopoietic organs studied (Fig. 3).Despite proliferative and differentiative potential of fetal thymus–derived CD34CD38 cells in the T-lymphopoiesis system, myeloid-favoring cultures yielded few cells. Thymus and spleen CD34CD38 cells produced only rare colonies. Regarding the lineage yield, the colony morphology of CFC derived from liver, bone marrow, and fetal blood all included erythroid, granulocyte-monocyte, and mixed cell type. However, the colony production by liver and bone marrow was predominated by erythroid BFU-E colonies (liver: 76.1 6.5%; bone marrow: 69.5 8.0%) consistent with the marked erythrogenic needs of the developing organism.

Table 2. In vitro T-lymphopoietic capacity of 5000 sorted fetal hematopoietic CD34CD38 cells: 16–22 weeks gestation (n  3)

T-Cell markers CD3CD4 CD3CD8 CD4CD8 Myelomonocytic markers CD33CD14 CD45 cell count t0  5000

Liver d14 (percent CD45) mean SEM

Blood d14 (percent CD45) mean SEM

Bone marrow d14 (percent CD45) mean SEM

Spleen d14 (percent CD45) mean SEM

24.5% 18.1 2.5% 4.3 0.5% 2.1 0.4%

13.4% 12.1 1.8% 0.52 0.5% 0.8 0.1%

15.0% 10.9 1.5% 1.6 0.3% 2.5 0.3%

1.3% 0.18 0.2% 0.48 0.1% 0.62 0.1%

39.5 3.5%

62.1 5.6%

39.2 3.7%

58,000 7215

69,000 8235

75,000 9800

Thymus d14 (percent CD45) mean SEM 81.3% 58.1 3.9% 12.1 2.4% 11.1 1.2%

ND

0.43 0.1%

10,100 1100

95,000 12,250

Sorted CD34CD38 cells from fetal liver, blood, bone marrow, spleen, and thymus were cocultured with murine thymic stroma for 14 days. The proportion of CD45 human cells ( SEM) that had the CD3CD4, CD3CD8, and CD4CD8 immunophenotype supporting T-cell differentiation and CD45 myelomonocytic cells bearing CD14 and CD33 that were generated in the coculture are shown. The results of three independent experiments are included. ND  not determined.

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Function associated with a primitive phenotype was assessed by LTC-IC using dilutions of sorted fetal liver, bone marrow, peripheral blood, thymus, and spleen CD34CD38 cells. A high frequency of LTC-IC was detected in fetal liver (642 32 per 100,000 cells) and bone marrow (480 65 per 100,000 cells) (Fig. 4). Fetal peripheral blood CD34CD38 cells had the highest of all detected LTC-IC frequency at 2095 per 100,000 cells, possibly reflecting the vigorous relocation of stem cells occurring in the second trimester. Of note, low or undetectable levels of LTC-IC were

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seen for fetal thymus (20 8 per 100,000 cells) and spleen (3 per 100,000 cells) (Fig. 4). The poor performance of the cells derived from these organs indicates that moving the cells to a myeloid-favoring stromal context does not revert the cells to myeloid capability.

Discussion Second-trimester gestation in the human is a unique ontogenic period in the extent of stem-cell migration and provides

Figure 2. CD34CD38 generate varied T-cell output depending on the tissue of origin. (A) The scatter plot (side scatter: SSC; forward scatter: FSC) and immunophenotype of cells generated from murine thymic stroma alone is shown. No cells staining for CD3, CD4, or CD8 were detected at day 14 of the culture. (B) Scatter plot of cells generated by the T-lymphopoiesis system from CD34CD38 input cells. (C) The flow cytometric immunophenotype of cells generated from human fetal liver, bone marrow, blood, spleen, and thymus CD34CD38 cells cocultured with murine thymic stroma for 14 days. Both double-positive (CD4CD8) T cells and single-positive (CD3CD4) T cells were generated from liver, blood, and thymic CD34CD38 cells. FACS isotype markers are placed at different positions in this figure because the data were generated in independent experiments with gates set independently based on isotype antibody staining. (D) CD3 T cells generated in the coculture system were harvested at day 7 or day 14 and exposed to medium alone or medium with added PHA and Con A. Tritiated thymidine uptake into these cells was then determined at day 1 following exposure to PHA and Con A. The mean and SEM of three independent experiments are shown.

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Figure 2. Continued.

an opportunity to assess immunophenotypically similar subsets of cells that are either in transition or recently resident in hematopoietic organs. Comparing them in individuals provides a description of the components of hematopoiesis in the developing human that is novel, to our knowledge. The prevalence of particular subsets of cells in the tissues yielded some unanticipated results, including the abundance of CD34 cells in the spleen and of CD56 cells in the blood. In addition, it is evident that each tissue is unique; that bone marrow is quite distinct from fetal liver, with a lower prevalence of CD71 cells and CD34CD38 cells consistent with different functions of the organs. No gross trends due to age of the fetus were evident though our sample number was small, precluding more detailed analysis. We have designed coculture systems for T-cell generation from lineage-negative CD34 cells [20,21]. Recently we have identified an inorganic polymer matrix of fixed pore size that has been shown by others to be useful both for the maintenance of primitive populations of bone marrow cells and for gene transduction of these cells with retroviral vectors [22,23]. T-cell maturation from CD2-depleted CD34 cells, CD34CD38 cells, or AC133 cells occurs when cultured on murine thymic feeder layers on this matrix without supplemental cytokines. Immature double-positive cells (CD4CD8) and single-positive (CD3CD4 and CD3CD8) T cells with a high proportion of T-cell receptor rearrangement excision circles were generated [16]. To date we have not been able to use this system for singlecell analysis, however, which limits the extent of conclusions that can be made about the precise lymphoid lineage potential of the CD2CD34CD38 cell population. The ability to place cells in either a lymphoid or myeloid differentiation context provided us with the opportunity to test the functional capability of immunophenotypically similar cells from the same individual, but from distinct tissue sources. We noted that lineage yield, as defined by the productive ca-

pacity of primitive cells within a given lineage assay, was variable among the CD34CD38 cells tested. The ability of cells from fetal liver to yield myeloid CFC was distinct from any other tissue source with a substantially higher output per input cell number. Therefore, comparable immunophenotypic cells have markedly different physiologic responses depending upon the tissue from which they were harvested. Similarly, we noted that the multipotentiality of primitive CD34CD38 cells was not preserved and cells had unique profiles of capability determined by the organ from which they were derived. This was most striking in the thymus-derived cells where abundant T-lymphoid production potential was evident from small numbers of input cells. Preferential production of T cells by primitive cells from thymus as opposed to liver, bone marrow, blood, or spleen likely reflects a true difference in T-lymphoid potential, but it cannot be excluded that the system we used favors only those cells already moving toward a T-lymphoid outcome. It is possible that the most uncommitted cells in the continuum of differentiation do not perform in our assay and are thereby underestimated.

Figure 3. CFC-forming capacity for human fetal hematopoietic CD34 CD38 cells. CFC assays were performed on sorted CD34CD38 cells from fetal liver, blood, bone marrow, spleen, and thymus. Erythroid and myeloid colonies were counted and the frequency of colonies (CFC count per 100 CD34CD38 cells SEM) determined at 14 days. The results of three independent experiments are shown.

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Figure 4. LTC-IC capability of CD34CD38 cells varies with the tissue of origin. Limiting dilution analysis of LTC-IC was performed using CD34CD38 input cells from the indicated tissue sources. The frequency of LTC-IC in the starting cell suspension was calculated using Poisson statistics as the reciprocal of the concentration of test cells that gave 37% negative cultures. Results from one of two experiments with comparable outcomes are shown.

Concurrent analysis of the primitive cells in a context mimicking bone marrow (LTC-IC) demonstrated their limited ability to respond to an alternative differentiation environment. The cells performed only in the context of the in vitro model of the tissue from which they were harvested. However, similar to the limitations of the T-lymphopoiesis system, the LTC-IC system is only a surrogate for stem-cell activity and, therefore, cells performing poorly in the assay may be either too mature or too immature to respond to the in vitro culture conditions. While we regard the CD34CD38 populations yielding low LTC-IC as most likely reflective of a cell pool with fewer stem cells and restricted differentiation potential, it remains possible that a true stem-cell population went undetected in the assay. To the extent that LTC-IC does reflect a stem-cell population capable of responding to myeloid differentiation cues in conditions similar to that of the bone marrow, irreversible lineage restriction appears to occur within the CD34CD38 compartment. The different ability of primitive cells to function in lineage-specific assay systems defined their potential. Our data do not exclude low-abundance multipotential cells in each organ, but argue against this given the negligible ability of

spleen CD34CD38 cells to yield T cells and the minimal LTC-IC output of thymic CD34CD38 cells. Rather, primitive populations have distinct, organ-specific potential favoring a model in which stem cells are not retained as fully multipotent cells in the secondary organs of hematopoiesis. Defining low-frequency multipotential cells would require detailed single-cell characterization that is not feasible given the constraints of the T-lymphopoiesis system. However, the data provide a human correlate to corresponding findings in the murine system by Kawamoto and colleagues, where they could not identify multipotential cells in the murine thymus [18], and to Res et al., who found an absence of the stem-cell marker, Thy-1, on primitive thymocytes [24]. While Res et al. noted T, NK, and dendritic potential in fetal thymic CD34CD38dim cells, it has been hypothesized that stem cells either 1) preferentially migrate based on preset lineage potential, only entering microenvironments where their lineage outcome is favored, 2) stochastically enter organs of differentiation and are immediately modified by the environmental context, restricting their potential to outcomes specified by their site of residence, or 3) migrate broadly, engrafting multiple tissues that favor one outcome or another, but maintaining stem-

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cell characteristics. The data presented here demonstrate fixed irreversible lineage restriction when primitive cells are resident in a hematopoietic organ, thereby excluding the third possibility. Whether primitive cells lose plasticity when emerging from the fetal liver or are transmuted upon arrival at the differentiation site cannot be defined by our data. However, the distinctions between fetal liver and their transport mechanism, the blood, suggest that the emigration phenomenon itself may involve selectivity. High-efficiency cell labeling, cell recovery, and sequential transplant may permit functional definition of this issue [25]. Acknowledgments The authors thank Drs. Michael Rosenzweig and Mark Pykett of Cytomatrix for their input and generous provision of CellFoam and Dr. Mark Van Gorder and Ms. Kathleen Sirois from the Department of Pathology at Brigham and Women’s Hospital, Boston, MA, for assistance in obtaining tissue. Support for this work was provided by the National Institutes of Health (D.T.S. and T.C.), the American Foundation for AIDS Research (M.P.), and the Richard Saltonstall Charitable Foundation (D.T.S.).

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