Rajesh Deepika, Chinnasamy Nachimuthu, Mitalipov Shoukhrat M, Wolf Don P, Slukvin Igor, Thomson James A, Shaaban Aimen F
Department of Surgery, University of Wisconsin Medical School, K4/760 Clinical Science Center, 600 Highland Avenue, Madison, Wisconsin 53792-7375, USA.
Stem Cells. 2007 Feb;25(2):490-9. doi: 10.1634/stemcells.2006-0277.
Progress toward clinical application of ESC-derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model. However, in contrast to human ESCs (hESCs), efforts to induce conclusive hematopoietic differentiation from rhesus macaque ESCs (rESCs) have been unsuccessful. Characterizing these poorly understood functional differences will facilitate progress in this area and likely clarify the critical steps involved in the hematopoietic differentiation of ESCs. To accomplish this goal, we compared the hematopoietic differentiation of hESCs with that of rESCs in both EB culture and stroma coculture. Initially, undifferentiated rESCs and hESCs were adapted to growth on Matrigel without a change in their phenotype or karyotype. Subsequent differentiation of rESCs in OP9 stroma led to the development of CD34(+)CD45(-) cells that gave rise to endothelial cell networks in methylcellulose culture. In the same conditions, hESCs exhibited convincing hematopoietic differentiation. In cytokine-supplemented EB culture, rESCs demonstrated improved hematopoietic differentiation with higher levels of CD34(+) and detectable levels of CD45(+) cells. However, these levels remained dramatically lower than those for hESCs in identical culture conditions. Subsequent plating of cytokine-supplemented rhesus EBs in methylcellulose culture led to the formation of mixed colonies of erythroid, myeloid, and endothelial cells, confirming the existence of bipotential hematoendothelial progenitors in the cytokine-supplemented EB cultures. Evaluation of four different rESC lines confirmed the validity of these disparities. Although rESCs have the potential for hematopoietic differentiation, they exhibit a pause at the hemangioblast stage of hematopoietic development in culture conditions developed for hESCs.
胚胎干细胞（ESC）来源的造血细胞移植在临床应用方面取得进展需要在大型动物同种异体模型中进行严格评估。然而，与人类胚胎干细胞（hESC）不同，从恒河猴胚胎干细胞（rESC）诱导出确定性造血分化的努力一直未成功。明确这些了解甚少的功能差异将有助于该领域的进展，并可能阐明胚胎干细胞造血分化所涉及的关键步骤。为实现这一目标，我们在胚体（EB）培养和基质共培养中比较了hESC和rESC的造血分化。最初，未分化的rESC和hESC适应在基质胶上生长，其表型和核型均未改变。随后，rESC在OP9基质中分化产生了CD34(+)CD45(-)细胞，这些细胞在甲基纤维素培养中形成了内皮细胞网络。在相同条件下，hESC表现出令人信服的造血分化。在添加细胞因子的EB培养中，rESC的造血分化有所改善，CD34(+)细胞水平更高，且可检测到CD45(+)细胞水平。然而，这些水平仍显著低于相同培养条件下hESC的水平。随后将添加细胞因子的恒河猴EB接种到甲基纤维素培养中，导致形成了红细胞、髓细胞和内皮细胞的混合集落，证实了添加细胞因子的EB培养中存在双能造血内皮祖细胞。对四种不同rESC系的评估证实了这些差异的有效性。尽管rESC具有造血分化的潜力，但在为hESC开发的培养条件下，它们在造血发育的成血管细胞阶段出现停滞。
